This work was supported by NIH Grant EY12155, EY027193, and EY027387 to JC. We are thankful to Dr. Spyridon Michalakis (Caltech, Pasadena, USA) and Natalie Chen (USC, Los Angeles, USA) for proofreading the manuscript. We would also like to thank Dr. Seth Ruffins (USC, Los Angeles, USA) and Dr. Janos Peti-Peterdi (USC, Los Angeles, USA) for providing the necessary equipment to collect the author provided footage. Material from: Kasey Rose et al, Separation of photoreceptor cell compartments in mouse retina for protein analysis, Molecular Neurodegeneration, published [2017], [Springer Nature].

All experiments were performed according to the local institutional guidelines of the committee on research animal care from the University of Southern California (USC).
1. Live cell retinal peeling method
<ol>
	<li>Preparation of Ames&#39; buffers, peeling papers, and dissection dish
	<ol>
		<li>Using blunt tip iris scissors (or equivalent scissor type), cut the cellulose filter paper (Grade 413) into rectangles measuring approximately 5 mm x 2.5 mm. Store the cuttings for future use.</li>
		<li>Prepare 1 L of Ames&#39; HEPES buffer by combining one bottle of Ames&#39; Medium, 2.38 g HEPES, and 0.877 g of NaCl. Dissolve the reagents in a sterile glass bottle with distilled ultrapure water and adjust the osmolarity and pH to 280 mOsm and 7.4, respectively. Filter to sterilize (pore size 0.2 &#181;m), seal the lid with paraffin film, and store at 4 &#176;C.</li>
		<li>Prepare 1 L of Ames&#39; bicarbonate buffer by combining one bottle of Ames&#39; Medium and 1.9 g of NaHCO3. Dissolve the reagents in a sterile glass bottle with distilled ultrapure water and adjust the osmolarity and pH to 280 mOsm and 7.4, respectively. Filter to sterilize (pore size 0.2 &#181;m), seal the lid with paraffin film, and store at 4 &#176;C. 
		NOTE: Ames&#39; HEPES and Ames&#39; bicarbonate buffers can be prepared in advance and stored for 1 week at 4 &#176;C.</li>
		<li>At the bottom of a 35 x 10 mm Petri dish, create a lattice or checkerboard pattern with a scalpel (blade No. 11, 40 mm). 
		NOTE: The textured bottom of the Petri dish holds the isolated, free-floating eye in place during the retinal dissection.</li>
	</ol>
	</li>
	<li>Dissection of the retina 
	NOTE: For this procedure, the buffer should be brought to and kept at room temperature (RT). Refresh the Ames&#39; bicarbonate buffer every 15 min with fresh carbogenated Ames&#39; bicarbonate buffer during the dissection. This maintains the necessary physiologic pH.
	<ol>
		<li>Around 15-20 min before the dissection, oxygenate the Ames&#39; HEPES buffer in a 100 mL laboratory or media bottle equipped with a tubing cap adapter and bubble the Ames&#39; Bicarbonate buffer with 95% O2 and 5% CO2 in a tissue incubation chamber and in a 100 mL laboratory or media bottle equipped with a tubing cap adapter.</li>
		<li>Euthanize an equal number of both genders of C57BL/6J mice (2-3 months old) either by isoflurane inhalation or carbon dioxide (CO2) followed by cervical dislocation. Enucleate the eyes with curved scissors and place the eyes into the textured 35 x 10 mm Petri dish filled with bubbled Ames&#39; bicarbonate buffer.</li>
		<li>Create an eye cup (Figure 1A) by puncturing a hole at the cornea-limbus junction with an 18 G needle. Cut along the perimeter of this junction using micro-dissection iris scissors to remove the cornea of each eye. Separate the lens and vitreous humor from each eye using tweezers and discard.</li>
		<li>Isolate the retina from the eye cup by carefully peeling the retinal pigmented epithelium (RPE)-sclera-choroid complex away from the neural retina. Discard the RPE-sclera-choroid complex. Ensure that the retina is not damaged during the dissection by handling only the edges.</li>
		<li>Transfer isolated retinae using a wide bore transfer pipette into the lid of a 60 x 15 mm dish filled with bubbled Ames&#39; bicarbonate buffer.</li>
		<li>Orient the hemisphere-shaped retina concave up (the photoreceptors are facing down toward the bottom of the dish) and bisect each retina (through the optic nerve) using iris scissors or a scalpel (blade No. 10, 40 mm). Trim the curved edges of each half retina to make two rectangles (Figure 1A). 
		NOTE: Minimizing the curvature of each halved retina allows for the retina to flatten better in subsequent peeling steps and produces an accurate peel of the rod outer segment (ROS) layer.</li>
		<li>Store the halved retinae in the tissue incubation chamber that is continuously bubbled with 95% O2 and 5% CO2. 
		NOTE: Isolated retinae can be kept in the carbogenated Ames&#39; bicarbonate buffer for ~24 h.</li>
	</ol>
	</li>
	<li>Rod outer segment (ROS) collection by filter paper peeling 
	NOTE: Refresh the RT oxygenated Ames&#39; HEPES buffer every 15-20 min during the peeling process to maintain the necessary physiologic pH.
	<ol>
		<li>Transfer one retinal rectangle from the tissue chamber with a wide bore transfer pipette and a 5 mm x 2.5 mm piece of filter paper into a 35 x 10 mm Petri dish filled with Ames&#39; HEPES buffer bubbled with 100% O2.</li>
		<li>Orient the partitioned retina concave up and ensure the photoreceptors are facing down toward the bottom of the dish. Lightly grasp the sides of the halved retina with tweezers and move it on top of the filter paper. Once the retina is centered on the filter paper, move the filter paper upward so the halved retina touches the filter paper to create the ROS-to-filter paper adhesion (Figure 1B). 
		NOTE: Make sure that the photoreceptor layer makes direct contact with the filter paper.</li>
		<li>Carefully lift the filter paper with the attached retina out of the Ames&#39; HEPES buffer. Place the bottom side of the filter paper (the side without the retina) on a paper towel and dab 2-3 times to dry (Figure 1B).</li>
		<li>Add a drop of Ames&#39; HEPES onto the side of the filter paper with the retina. Place the filter paper on the paper towel again to dry, as just described. Repeat this process two more times.</li>
		<li>Place the filter paper with the retina back into the Petri dish. Using tweezers, gently push all edges of the halved retina up and away from the filter paper on all sides.</li>
		<li>Delicately peel the retina away from the filter paper. Touch only the extreme perimeter of the retina during the peeling process to preserve the retina&#39;s structural integrity.</li>
		<li>Lift the filter paper from the Petri dish and remove excess liquid on a paper towel. Make sure the side that was in contact with the retina does not contact the paper towel.</li>
		<li>Place the filter paper with the partial ROS layer into a labeled tube on ice (Figure 1B). Keep the peeled retina submerged in Ames&#39; HEPES buffer.</li>
		<li>Repeat the peeling process for this halved retina approximately 7-8 times to remove the entire ROS layer. After each peel, place the filter paper into the same tube, which will contain the isolated ROS from one halved retina. 
		&#8203;NOTE: Take care to not tear the halved retina when peeling it away from the filter paper, as the retina becomes thinner during this process.</li>
		<li>Place the tube containing all filter paper peels of the isolated ROS on ice for immediate use or freeze directly on dry ice and store at -80 &#176;C.</li>
		<li>Use tweezers to transfer the leftover peeled retina (lacking ROS) into an appropriately labeled tube. Place the tube on ice for immediate use or freeze with dry ice and store at -80 &#176;C.</li>
		<li>Repeat the peeling process for each halved retina and accurately label each tube.</li>
	</ol>
	</li>
</ol>
2. Lyophilized retina peeling method
<ol>
	<li>Buffers, peeling medium, dissection dish preparation, and lyophilizer set-up
	<ol>
		<li>In a 1 L storage bottle, prepare the Ringer&#39;s solution by adding 500 mL of distilled ultrapure water. Dissolve the reagents to reach a final concentration of 130 mM NaCl, 3.6 mM KCl, 2.4 mM MgCl2, 1.2 mM CaCl2, 10 mM HEPES, and 0.02 mM EDTA. Adjust the pH to 7.4 with NaOH, add ultrapure water to bring the final volume to 1 L, and adjust osmolarity to 313 mOsm.</li>
		<li>Prior to the use, filter the Ringer&#39;s solution with a sterile filter (pore size 0.2 &#181;m), seal the lid with paraffin film, and store at 4 &#176;C.</li>
		<li>Using blunt tip iris scissors (or equivalent scissor type), cut the cellulose filter paper into rectangles measuring approximately 5 mm x 2.5 mm. Use a pencil to write on one side of the filter paper to create a unique label.</li>
		<li>Create a lattice or checkerboard pattern in the bottom of a 35 x 10 mm Petri dish using a scalpel (blade No. 11, 40 mm).</li>
		<li>Turn on the lyophilizer according to the manufacturer&#39;s specifications.</li>
		<li>Acquire liquid nitrogen in a Dewar flask or similar insulating vacuum flask.</li>
	</ol>
	</li>
	<li>Retinal dissection
	<ol>
		<li>Complete the retinal dissection as described in section 1 of the protocol (Figure 1A). Use cold Ringer&#39;s solution instead of Ames&#39; buffer.</li>
		<li>Post dissection, store the halved, rectangular retinae in a 35 x 10 mm Petri dish filled with cold Ringer&#39;s solution and place on ice.</li>
	</ol>
	</li>
	<li>Retinal sample preparation for lyophilization
	<ol>
		<li>Place a 5 x 2.5 mm piece of filter paper and transfer a halved retina into the texturized 35 x 10 mm Petri dish filled with cold Ringer&#39;s buffer. Use a wide bore transfer pipette to move each retina between dishes.</li>
		<li>Use tweezers to carefully flip the retina so it is oriented concave up (the photoreceptors are pointing upward) and move it so it rests on top of the filter paper (Figure 2A). 
		NOTE: Make sure the photoreceptors do not contact the filter paper (the retinal ganglion cell layer should be in direct contact with the filter paper) and the unique label is visible. To easily and quickly identify the retinal isolate, it is recommended that the unique label should be located on the underside of the filter paper.</li>
		<li>Lift the filter paper by the edges upward so the halved piece of retina touches the filter paper and continue to lift the filter paper out of the Ringer&#39;s solution.</li>
		<li>Place the bottom side of the filter paper (the side without the retina on it) on a paper towel and carefully dab 2-3 times to remove excess liquid (Figure 2A).</li>
		<li>Pick up the filter paper with tweezers and add a drop of cold Ringer&#39;s onto the side of the filter paper with the retina. Place the filter paper on the paper towel again to remove excess liquid. Repeat the process two more times. 
		NOTE: These alternating wetting and drying steps ensure that the tissue is firmly attached to the filter paper.</li>
		<li>Store each adhered retina/filter paper sample in a Petri dish filled with cold Ringer&#39;s until ready to freeze all samples. Repeat this process for all halved retinae.</li>
		<li>Obtain a clean and dry 35 x 10 mm Petri dish (bottom only) and a piece of aluminum foil cut into a 3.5 x 3.5 inch square.</li>
		<li>Lift each sample out of the cold Ringer&#39;s buffer with tweezers. Ensure that the tweezers contact only the filter paper edges, avoiding the halved retina.</li>
		<li>Place the bottom side of each filter paper (the side without the retina on it) on a paper towel and remove excess liquid.</li>
		<li>Add a drop of cold 1x PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4) onto the side of the filter paper where the retina is adhered. Dab the filter paper on the paper towel to remove excess liquid. 
		NOTE: Make sure that the moisture from the filter paper is sufficiently removed before freezing.</li>
		<li>Place all filter paper pieces into the 35 x 10 mm Petri dish. Use a lint free tissue paper to wick away any excess liquid surrounding the filter paper. 
		NOTE: Do not overcrowd the Petri dish with samples. If more than four murine eyes are used, consider using a larger Petri dish or multiple smaller Petri dishes to hold the retina/filter paper samples.</li>
		<li>Tightly wrap the entire dish with aluminum foil. Ensure that the edges of the aluminum foil are secured and smoothly pressed into the bottom of the Petri dish. Puncture a handful of 0.1-0.2 mm holes into the aluminum foil lid (Figure 2A).</li>
		<li>Using metal tongs, gradually lower the aluminum foil covered Petri dish into the liquid nitrogen. Keep the samples in the liquid nitrogen (&#60;10 min) until ready to lyophilize.</li>
	</ol>
	</li>
	<li>Lyophilization
	<ol>
		<li>Place the aluminum foil covered Petri dish into a freeze-drying flask and attach it to the lyophilizer machine following the manufacturer&#39;s protocol. Lyophilize the retinae for 30 min.</li>
		<li>Detach the flask from the lyophilizer and shut off the machine according to the manufacturer.</li>
		<li>Remove the aluminum foil and either work with the freeze-dried samples the same day or store them in properly labeled 1 mL microcentrifuge tubes. Place these tubes in a container (such as a 50 mL conical tube) filled with an anhydrous desiccant and store the samples at -80 &#176;C.</li>
	</ol>
	</li>
	<li>Rod outer and inner segment collection by adhesive tape peeling.
	<ol>
		<li>Cut strips of clear adhesive tape (1-2 cm) and store strips on the edge of the tape dispenser/lab bench/etc. for quick use.</li>
		<li>Move one retinal sample to the lid of a plastic 60 x 15 mm Petri dish for peeling. Ensure to only grab the outermost edges of the filter paper and avoid touching the retina.</li>
		<li>Using blunt tip iris scissors (or equivalent scissor type), cut a small rectangular piece of tape to the approximate size of the tissue (1.5 x 2.5 mm).</li>
		<li>Carefully lay a small piece of tape on top of the lyophilized retina (Figure 2B) and apply slight pressure with the tweezers to make sure it bonds with the photoreceptor layer (the orange/pink-tinted top layer).</li>
		<li>Slowly peel the tape away. Both the rod outer segment (ROS) and rod inner segment (RIS) will be adhered to the tape (Figure 2B).</li>
		<li>Ensure that there is a thin white film at the fractured surface. This is RIS.To separate the RIS, place another piece of tape and push the tape down on the fractured surface (Figure 2B). 
		NOTE: It may take more than one piece of tape to remove the entire thin white layer.</li>
		<li>Place the piece of tape with only the orange/pink layer into a microcentrifuge tube labeled +ROS.</li>
		<li>Place the tape or pieces of tape with the thin white layer into a microcentrifuge tube labeled +RIS. 
		NOTE: Separating the lyophilized retina with tape takes practice. The amount of pressure applied to the tape will affect how the lyophilized sample will fraction. If too much pressure is placed on the tape over the sample, the whole lyophilized retina will peel off the filter paper and will stick to the tape. If too little pressure is added to the tape, the orange/pink top layer (+ROS) will not stick to the tape.</li>
		<li>Collect the leftover retinal tissue (bereft of ROS and RIS layers, -OIS) located on the filter paper by peeling off the thick, white layer from the filter paper using tape. Place the isolate into a tube labeled -OIS.</li>
		<li>Repeat these steps for each halved retinal sample.</li>
		<li>Store the isolated layers from the lyophilized retinae either at room temperature if performing protein quantification, gel electrophoresis, and protein immunoblots that same day, or store in a container (such as 50 mL conical tube) filled with anhydrous desiccant at -80 &#176;C for later use.</li>
	</ol>
	</li>
</ol>
3. Western blot sample preparation for peeling isolations
<ol>
	<li>Prepare RIPA lysis buffer
	<ol>
		<li>In a 100 mL storage bottle, add reagents to obtain a final concentration of 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% Sodium deoxycholate, 0.1% SDS, and 1 mM EDTA. Add 100 mL of deionized ultrapure water and mix thoroughly. 
		NOTE: RIPA lysis buffer can be stored at 2-8 &#176;C for 2-3 weeks. For longer storage, aliquot and store in the -20 &#176;C freezer.</li>
		<li>On the day of the experiment, add a protease inhibitor cocktail (0.1 M PMSF, 1:1000 Aprotinin, and 1:1000 Leupeptin) to the RIPA lysis buffer and keep on ice.</li>
	</ol>
	</li>
	<li>Filter paper peeling lysate preparation for western blot
	<ol>
		<li>Ensure that tubes containing the filter paper peels and leftover peeled retinae are maintained on ice (for the experiment on the same day) or removed from -80 &#176;C freezer storage and placed on ice.</li>
		<li>Remove any excess liquid from the bottom of the microcentrifuge tubes containing the filter paper peels. Quickly spin in a mini centrifuge for 2-4 s and discard any remaining liquid.</li>
		<li>Pipette 45-55 &#181;L of cold RIPA buffer into tubes containing the filter paper isolate.</li>
		<li>Homogenize each sample with a clean, autoclaved pestle mixer for 1 min. Make sure the filter paper stays in contact with the pestle mixer. Keep the filter paper on the side of each tube rather than at the bottom.</li>
		<li>With tweezers, move the filter papers to the side of each tube and spin in the mini centrifuge for 2-4 s. This will remove the RIPA buffer from the filter paper.</li>
		<li>Remove dried filter paper and repeat for all filter papers within each tube if necessary.</li>
		<li>Transfer the homogenate to a new tube and label it appropriately. 
		NOTE: This is the most time-consuming step, and if not completed properly, much of the sample will end up being absorbed by the filter paper.</li>
		<li>Pipette 60-80 &#181;L of cold RIPA buffer into the individual tubes with the leftover peeled retinae.</li>
		<li>Homogenize each sample with a clean, autoclaved pestle mixer for 1 min.</li>
	</ol>
	</li>
	<li>Tape peeling Lysate preparation for western blot
	<ol>
		<li>Place the RT lyophilized samples or the -80 &#176;C samples on ice.</li>
		<li>Pipette 50-70 &#181;L of cold RIPA buffer into each tube containing +ROS and +RIS tape peels.</li>
		<li>Pipette 60-80 &#181;L of cold lysis buffer into the -OIS tubes containing the remaining lyophilized retina, with the ROS and RIS removed.</li>
		<li>Homogenize each sample with a clean pestle mixer for 1 min. Ensure that the tape in the individual tubes stays in contact with the pestle mixer and lysis buffer.</li>
		<li>With sterilized tweezers, move the tape to the side of the tubes and spin with a mini centrifuge for 2-4 s. This will remove the liquid from the tape. Remove the dried tape from the tubes. 
		NOTE: At this point, you may combine two half retinal isolates from the same retina for either the &#39;filter paper peeling&#39; or &#39;tape peeling&#39; method to ensure you have enough volume to perform a bicinchoninic acid assay (BCA).</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Arshavsky, V. Y., Lamb, T. D., Pugh, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=G+proteins+and+phototransduction.">G proteins and phototransduction.</a> Annual Review of Physiology. 64, 153-187 (2002).</li><li>Molday, R. S., Moritz, O. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoreceptors+at+a+glance.">Photoreceptors at a glance.</a> Journal of Cell Science. 128 (22), 4039-4045 (2015).</li><li>Koch, K. W., Dell'Orco, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+and+signaling+networks+in+vertebrate+photoreceptor+cells.">Protein and signaling networks in vertebrate photoreceptor cells.</a> Frontiers in Molecular Neuroscience. 8, 67 (2015).</li><li>Semple-Rowland, S. L., Dawson, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic+light+intensity+threshold+for+retinal+damage+in+albino+rats+raised+under+6+lx.">Cyclic light intensity threshold for retinal damage in albino rats raised under 6 lx.</a> Experimental Eye Research. 44 (5), 643-661 (1987).</li><li>Brann, M. R., Cohen, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diurnal+expression+of+transducin+mRNA+and+translocation+of+transducin+in+rods+of+rat+retina.">Diurnal expression of transducin mRNA and translocation of transducin in rods of rat retina.</a> Science. 235 (4788), 585-587 (1987).</li><li>Philp, N. J., Chang, W., Long, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-stimulated+protein+movement+in+rod+photoreceptor+cells+of+the+rat+retina.">Light-stimulated protein movement in rod photoreceptor cells of the rat retina.</a> FEBS Letters. 225 (1-2), 127-132 (1987).</li><li>Broekhuyse, R. M., Tolhuizen, E. F., Janssen, A. P., Winkens, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light+induced+shift+and+binding+of+S-antigen+in+retinal+rods.">Light induced shift and binding of S-antigen in retinal rods.</a> Current Eye Research. 4 (5), 613-618 (1985).</li><li>Roof, D. J., Heth, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+transducin+in+retinal+rod+photoreceptor+outer+segments.">Expression of transducin in retinal rod photoreceptor outer segments.</a> Science. 241 (4867), 845-847 (1988).</li><li>Sokolov, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Massive+light-driven+translocation+of+transducin+between+the+two+major+compartments+of+rod+cells:+a+novel+mechanism+of+light+adaptation.">Massive light-driven translocation of transducin between the two major compartments of rod cells: a novel mechanism of light adaptation.</a> Neuron. 34 (1), 95-106 (2002).</li><li>Lobanova, E. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transducin+translocation+in+rods+is+triggered+by+saturation+of+the+GTPase-activating+complex.">Transducin translocation in rods is triggered by saturation of the GTPase-activating complex.</a> The Journal of Neuroscience. 27 (5), 1151-1160 (2007).</li><li>Strissel, K. J., Sokolov, M., Trieu, L. H., Arshavsky, V. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arrestin+translocation+is+induced+at+a+critical+threshold+of+visual+signaling+and+is+superstoichiometric+to+bleached+rhodopsin.">Arrestin translocation is induced at a critical threshold of visual signaling and is superstoichiometric to bleached rhodopsin.</a> The Journal of Neuroscience. 26 (4), 1146-1153 (2006).</li><li>Nair, K. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-dependent+redistribution+of+arrestin+in+vertebrate+rods+is+an+energy-independent+process+governed+by+protein-protein+interactions.">Light-dependent redistribution of arrestin in vertebrate rods is an energy-independent process governed by protein-protein interactions.</a> Neuron. 46 (4), 555-567 (2005).</li><li>Strissel, K. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recoverin+undergoes+light-dependent+intracellular+translocation+in+rod+photoreceptors.">Recoverin undergoes light-dependent intracellular translocation in rod photoreceptors.</a> The Journal of Biological Chemistry. 280 (32), 29250-29255 (2005).</li><li>Calvert, P. D., Strissel, K. J., Schiesser, W. E., Pugh, E. N., Arshavsky, V. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-driven+translocation+of+signaling+proteins+in+vertebrate+photoreceptors.">Light-driven translocation of signaling proteins in vertebrate photoreceptors.</a> Trends in Cell Biology. 16 (11), 560-568 (2006).</li><li>Majumder, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transducin+translocation+contributes+to+rod+survival+and+enhances+synaptic+transmission+from+rods+to+rod+bipolar+cells.">Transducin translocation contributes to rod survival and enhances synaptic transmission from rods to rod bipolar cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (30), 12468-12473 (2013).</li><li>Fain, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Why+photoreceptors+die+(and+why+they+don't).">Why photoreceptors die (and why they don't).</a> BioEssays. 28 (4), 344-354 (2006).</li><li>Chen, J., Simon, M. I., Matthes, M. T., Yasumura, D., LaVail, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+susceptibility+to+light+damage+in+an+arrestin+knockout+mouse+model+of+Oguchi+disease+(stationary+night+blindness).">Increased susceptibility to light damage in an arrestin knockout mouse model of Oguchi disease (stationary night blindness).</a> Investigative Ophthalmology &amp; Visual Science. 40 (12), 2978-2982 (1999).</li><li>Song, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arrestin-1+expression+level+in+rods:+balancing+functional+performance+and+photoreceptor+health.">Arrestin-1 expression level in rods: balancing functional performance and photoreceptor health.</a> Neuroscience. 174, 37-49 (2011).</li><li>McConnell, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+isolation+of+retinal+outer+segment+fragments.">The isolation of retinal outer segment fragments.</a> The Journal of Cell Biology. 27 (3), 459-473 (1965).</li><li>Tsang, S. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+for+the+target+enzyme+in+deactivation+of+photoreceptor+G+protein+in+vivo.">Role for the target enzyme in deactivation of photoreceptor G protein in vivo.</a> Science. 282 (5386), 117-121 (1998).</li><li>Walston, S. T., Chow, R. H., Weiland, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patch+clamp+recordings+of+retinal+bipolar+cells+in+response+to+extracellular+electrical+stimulation+in+wholemount+mouse+retina.">Patch clamp recordings of retinal bipolar cells in response to extracellular electrical stimulation in wholemount mouse retina.</a> Annual International Conference of the IEEE Engineering in Medicine and Biology Society. 2015, 3363-3366 (2015).</li><li>Guido, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+to+obtain+retinal+cell+preparations+highly+enriched+in+specific+cell+types.+Suitability+for+lipid+metabolism+studies.">A simple method to obtain retinal cell preparations highly enriched in specific cell types. Suitability for lipid metabolism studies.</a> Brain Research. Brain Research Protocols. 4 (2), 147-155 (1999).</li><li>Hayashi, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorylation+by+cyclin-dependent+protein+kinase+5+of+the+regulatory+subunit+of+retinal+cGMP+phosphodiesterase.+II.+Its+role+in+the+turnoff+of+phosphodiesterase+in+vivo.">Phosphorylation by cyclin-dependent protein kinase 5 of the regulatory subunit of retinal cGMP phosphodiesterase. II. Its role in the turnoff of phosphodiesterase in vivo.</a> The Journal of Biological Chemistry. 275 (42), 32958-32965 (2000).</li><li>Wolbring, G., Cook, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+purification+and+characterization+of+protein+kinase+C+from+bovine+retinal+rod+outer+segments.">Rapid purification and characterization of protein kinase C from bovine retinal rod outer segments.</a> European Journal of Biochemistry. 201 (3), 601-606 (1991).</li><li>Williams, D. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+protein+kinase+C+in+photoreceptor+outer+segments.">Characterization of protein kinase C in photoreceptor outer segments.</a> Journal of Neurochemistry. 69 (4), 1693-1702 (1997).</li><li>Watson, A. J., Aragay, A. M., Slepak, V. Z., Simon, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+form+of+the+G+protein+beta+subunit+Gbeta5+is+specifically+expressed+in+the+vertebrate+retina.">A novel form of the G protein beta subunit Gbeta5 is specifically expressed in the vertebrate retina.</a> The Journal of Biological Chemistry. 271 (45), 28154-28160 (1996).</li><li>Makino, E. R., Handy, J. W., Li, T., Arshavsky, V. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+GTPase+activating+factor+for+transducin+in+rod+photoreceptors+is+the+complex+between+RGS9+and+type+5+G+protein+beta+subunit.">The GTPase activating factor for transducin in rod photoreceptors is the complex between RGS9 and type 5 G protein beta subunit.</a> Proceedings of the National Academy of Sciences of the United States of America. 96 (5), 1947-1952 (1999).</li><li>Chen, J., Shi, G., Concepcion, F. A., Xie, G., Oprian, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+rhodopsin/arrestin+complex+leads+to+retinal+degeneration+in+a+transgenic+mouse+model+of+autosomal+dominant+retinitis+pigmentosa.">Stable rhodopsin/arrestin complex leads to retinal degeneration in a transgenic mouse model of autosomal dominant retinitis pigmentosa.</a> The Journal of Neuroscience. 26 (46), 11929-11937 (2006).</li><li>Chen, C. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abnormal+photoresponses+and+light-induced+apoptosis+in+rods+lacking+rhodopsin+kinase.">Abnormal photoresponses and light-induced apoptosis in rods lacking rhodopsin kinase.</a> Proceedings of the National Academy of Sciences of the United States of America. 96 (7), 3718-3722 (1999).</li><li>Zhang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mistrafficking+of+prenylated+proteins+causes+retinitis+pigmentosa+2.">Mistrafficking of prenylated proteins causes retinitis pigmentosa 2.</a> FASEB Journal. 29 (3), 932-942 (2015).</li><li>Weiss, E. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Species-specific+differences+in+expression+of+G-protein-coupled+receptor+kinase+(GRK)+7+and+GRK1+in+mammalian+cone+photoreceptor+cells:+implications+for+cone+cell+phototransduction.">Species-specific differences in expression of G-protein-coupled receptor kinase (GRK) 7 and GRK1 in mammalian cone photoreceptor cells: implications for cone cell phototransduction.</a> The Journal of Neuroscience. 21 (23), 9175-9184 (2001).</li><li>Zhao, X., Huang, J., Khani, S. C., Palczewski, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+forms+of+human+rhodopsin+kinase+(GRK1).">Molecular forms of human rhodopsin kinase (GRK1).</a> The Journal of Biological Chemistry. 273 (9), 5124-5131 (1998).</li><li>Newton, A. C., Williams, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+protein+kinase+C+in+the+phosphorylation+of+rhodopsin.">Involvement of protein kinase C in the phosphorylation of rhodopsin.</a> The Journal of Biological Chemistry. 266 (27), 17725-17728 (1991).</li><li>Pinzon-Guzman, C., Zhang, S. S., Barnstable, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+protein+kinase+C+isoforms+are+required+for+rod+photoreceptor+differentiation.">Specific protein kinase C isoforms are required for rod photoreceptor differentiation.</a> The Journal of Neuroscience. 31 (50), 18606-18617 (2011).</li><li>Sokal, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+protein+kinase+C+isozymes+responsible+for+the+phosphorylation+of+photoreceptor-specific+RGS9-1+at+Ser475.">Identification of protein kinase C isozymes responsible for the phosphorylation of photoreceptor-specific RGS9-1 at Ser475.</a> The Journal of Biological Chemistry. 278 (10), 8316-8325 (2003).</li><li>Rose, K., Walston, S. T., Chen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+of+photoreceptor+cell+compartments+in+mouse+retina+for+protein+analysis.">Separation of photoreceptor cell compartments in mouse retina for protein analysis.</a> Molecular Neurodegeneration. 12 (1), 28 (2017).</li><li>Wright, A. F., Chakarova, C. F., Abd El-Aziz, M. M., Bhattacharya, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoreceptor+degeneration:+genetic+and+mechanistic+dissection+of+a+complex+trait.">Photoreceptor degeneration: genetic and mechanistic dissection of a complex trait.</a> Nature Reviews. Genetics. 11 (4), 273-284 (2010).</li></ol>

The authors declare that they have no competing interests.

Many retinal diseases affect rod photoreceptor cells, leading to rod death and, ultimately, complete vision loss37. A significant portion of the genetic and mechanistic origins of human retinal degeneration have been successfully recapitulated in numerous mouse models over the years. In that context, the ability to easily and selectively separate individual rod subcellular compartments from the small mouse retina would greatly enhance our understanding of the localized biochemical and molecular underpinnings of retinal diseases. Furthermore, the layered nature of the neural retina, combined with the unique, highly polarized arrangement of rod photoreceptors, permits the isolation of rod compartments by selective peeling. This manuscript describes two protocols used to isolate individual subcellular compartments of rod photoreceptor cells for immunoblotting. Not only are both methods considerably faster and less technically challenging when compared to the existing method of tangential sectioning9, but they also require a substantially smaller sample size than the common biochemical isolation of the ROS using density gradients19. Such ROS gradient purification techniques require 8-10 murine retinae to reliably recover ample ROS, whereas the protocols presented here are suitable for single retinae.
Despite the overall simplicity of the proposed techniques, one of the main challenges common to both protocols concerns the flattening of the curved retina onto the filter paper required for proper isolation of the different subcellular compartments. The isolated retina tends to curl up and take on a clam-like shape. If the retina has folded edges when placed on the filter paper (photoreceptor side either up or down), this may decrease the yield of the desired isolated rod layers. More importantly, if the retina is not properly flattened, then layer misalignment and contamination from other subcellular compartments and retinal cells is a likely outcome (Figure 6D). To limit the curvature of the halved retinal rectangle and to rectify the imperfect alignment of the rod and retinal layers, the retinal rectangle can be cut in half to produce two retinal squares. By cutting the neural retina into smaller pieces, the natural curvature of the retina is reduced, and the tissue is more likely to lay flat on the filter paper.
Recognizing when the different rod compartments have been physically separated can be tricky for someone inexperienced with these techniques. We advise that before commencing a biologically significant experiment, the user should run through the process with sample retinae for an hour or two. Practice should result in substantial improvement in the operator's skills and proficiency in the methods. When using filter paper to peel away the ROS from a live retina, no obvious visual cues indicate when the ROS layer has been isolated. While the retina becomes thinner and more fragile, the user will have to self-monitor and validate the number of peels to isolate the ROS accurately. Furthermore, using filter paper with different fiber thicknesses and coarseness will alter the ROS peeling time. Filter paper with thicker fibers (such as VWR Grade 413) will isolate retinal layers faster (6-8 peels), but may not be as selective and can lead to contamination from other layers. Filter paper with thinner fibers (such as Whatman Grade 1) will isolate retinal layers slower (12-15 peels) and will give a clean isolation of the photoreceptor layers. We recommend using both thicker and thinner filter paper to minimize the peeling time and ensure collection purity.
At the same time, visual approximation and tape sample handling are both key to the success of isolating the subcellular compartments from the lyophilized retina. If too much pressure is added to the tape, the entire lyophilized retina will adhere to the tape. Once this occurs, separating the ROS becomes lengthier and more difficult, but not impossible: place a second piece of tape on the free surface (on the ganglion cell side) of the retina and slowly pull away the layers with tape until only the orange/pinkish layer is visible on the initial piece of tape. Isolating the RIS at this point, however, is impossible. If too little pressure is added to the tape, a large portion of the ROS will not adhere to the tape. To ensure proper ROS-tape adhesion, we advise gently pressing with tweezers on the regions that are not sticking to the tape. Typically, the slightest pressure results in the removal of the ROS and RIS layers, however, the amount of pressure must be determined based on experience. The presence of isolated RIS can be ascertained by checking whether a thin, white layer (akin to a light dusting of snow) is visible on the initial ROS tape peel, while the presence of isolated ROS can be easily determined by checking the color of the rod photoreceptor layer (orange for dark-adapted, pinkish for light-adapted) on the tape.
One final challenge arises when homogenizing the filter paper peels (+ROS) from the live retina. Filter paper easily absorbs liquid, and a considerable amount of the homogenizing buffer will be soaked up during this step of the protocol. Spinning the liquid down in a mini centrifuge after placing the peeling paper on the side of each tube helps prevent the loss of sample. Unfortunately, spinning down multiple pieces of filter paper can be a time-consuming process, and loss of sample is inevitable. To address this issue, remove some of the filter paper pieces and focus on homogenizing fewer pieces at a time. Additionally, to avoid the loss of sample during this step, consider minimizing the total amount of filter paper used to isolate the ROS.
While both our methods do not require a large sample size, or the precise alignment of a blade to section the retina, there are a few limitations to our photoreceptor peeling techniques that are worth noting. First, the lyophilized murine retina may not be amenable for peeling subsequent photoreceptor layers beyond the ROS and RIS. Second, our peeling methods are more suited for processing individual retinae and are not amenable to large batch processing in one sitting. Third, the success of the experiment is dependent on the sensitivity limit of the antibody used in the western blot. Despite these limitations, our representative results demonstrate that our two isolation peeling techniques can efficiently isolate specific rod photoreceptor compartments. Additionally, these two methods use inexpensive and commonplace lab materials (filter paper and adhesive tape), making them highly accessible. In our study, we did not directly assess whether other retinal cells could be isolated for immunoblotting; however, we believe that these methods may be easily modified to benefit the needs of a broad cross-section of the retinal research community.

Rod photoreceptor cells, tightly packed in the outermost layer of the neural retina, are an integral part of dim light vision. To function as faithful photon counters, rods utilize a G-protein-based signaling pathway, termed phototransduction, to generate rapid, amplified, and reproducible responses to single photon capture. This response to light ultimately triggers a change in the current at the plasma membrane and is subsequently signaled to the rest of the visual system1. As their name implies, each rod cell has a distinct rod-like shape and exhibits a highly polarized cellular morphology, consisting of an outer segment (OS), inner segment (IS), cell body (CB), and synaptic terminal (ST). Each subcellular compartment has specific protein machinery (membrane-bound and soluble), biomolecular features, and protein complexes that play crucial roles such as visual phototransduction, general housekeeping and protein synthesis, and synaptic transmission2,3.
Over 30 years ago, the light-dependent reciprocal movement of subcellular proteins, specifically transducin (away from the OS) and arrestin (towards the OS), was first observed4,5,6,7. Early on, this observed phenomenon was received with skepticism, due in part to immunohistochemistry&#39;s vulnerability to epitope masking8. In the early 2000s, stimulus-dependent protein translocation was confirmed by using a rigorous and arduous physical sectioning technique9. Serial tangential sectioning of the frozen flat-mounted rodent retinae followed by immunoblotting revealed that transducin9,10, arrestin11,12, and recoverin13 all undergo subcellular redistribution in response to light. It is believed that light-driven translocation of these key signaling proteins not only regulates the sensitivity of the phototransduction cascade9,14,15, but may also be neuroprotective against light damage16,17,18. Because light-driven protein transport in rods appears to be very significant to rod cell biology and physiology, techniques that permit the isolation of different subcellular compartments to determine protein distribution are valuable research tools.
Currently, there are a few methods aimed at isolating the rod subcellular compartments. However, these methods can be lengthy and difficult to reproduce, or require a sizable amount of retinal isolate. Rod outer segment (ROS) preparations via density gradient centrifugation19, for example, is commonly used to separate the ROS from retinal homogenate. This method is widely used for western blot, but the procedure is very time consuming and requires a minimum of 8-12 murine retinae20. On the other hand, serial tangential sectioning of frozen murine and rat retinae has been successfully implemented in isolating the OS, IS, CB, and ST9,11,13. However, this method is technically challenging due to the necessity of fully flattening the small and highly curved murine retina to align the retinal layers prior to tangential sectioning. Since there are a plethora of mouse models and transgenic mice recapitulating diseases of the visual system, the creation of a technique that reliably, quickly, and easily separates individual rod compartments holds promise in revealing the physiologic processes that occur in each specialized compartment and the mechanisms that underlie visual processes in health and disease.
To facilitate these investigations, we describe two peeling methods that isolate rod subcellular compartments more easily than current protocols. The first peeling method, adapted from a technique to expose fluorescently labeled bipolar cells for patch clamp recording21, employs cellulose filter paper to sequentially remove the ROS from a live, isolated murine retina (Figure 1). The second method, adapted from a procedure that isolates the three primary retinal cell layers from a chick22 and frog23 retina, utilizes adhesive tape to remove the ROS and rod inner segment (RIS) from a lyophilized retina (Figure 2). Both procedures can be completed in 1 h and are considerably user-friendly. We provide validation of the effectiveness of these two separation protocols for western blot by utilizing dark-adapted and light-exposed retinae from C57BL/6J mice to demonstrate light-induced translocation of rod transducin (GNAT1) and arrestin (ARR1). Moreover, using the tape peeling method, we provide additional evidence that our technique can be used to examine and address inconsistencies between protein localization data acquired by immunocytochemistry (ICC) and western blots. Specifically, our technique showed that: 1) the protein kinase C-alpha (PKC&#945;) isoform is present not only in bipolar cells, but also in murine ROS and RIS, albeit in low concentrations24,25, and 2) rhodopsin kinase (GRK1) is present predominantly in the isolated OS sample. These data demonstrate the effectiveness of our two peeling techniques for separating and quantifying specific rod and retinal proteins.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>100 mL laboratory or media bottle equipped with a tubing cap adapter</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>100% O2 tank</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>1000mL Bottle Top Filter, PES Filter Material, 0.22 &#956;m</td>
			<td>Genesee Scientific</td>
			<td>25-235</td>
			<td></td>
		</tr>
		<tr>
			<td>4X SDS Sample Buffer</td>
			<td>Millipore Sigma</td>
			<td>70607-3</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Falcon tube</td>
			<td>Fisher Scientific</td>
			<td>14-432-22</td>
			<td></td>
		</tr>
		<tr>
			<td>95% O2 and 5% CO2 tank</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Ames&#8217; Medium with L-glutamine, without bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>A1420</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2 (99%, dihydrate)</td>
			<td>Sigma</td>
			<td>C-3881</td>
			<td></td>
		</tr>
		<tr>
			<td>Drierite (Anhydrous calcium sulfate, &#62;98% CaSO4, &#62;2% CoCl2)</td>
			<td>WA Hammond Drierite Co LTD</td>
			<td>21005</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon Easy-Grip Petri Dish (polystyrene, 35 x 10 mm)</td>
			<td>Falcon-Corning</td>
			<td>08-757-100A</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon Easy-Grip Tissue Culture Dish (60 x 15 mm)</td>
			<td>Falcon-Corning</td>
			<td>08-772F</td>
			<td></td>
		</tr>
		<tr>
			<td>Feather Scalpel (No. 10, 40 mm)</td>
			<td>VWR</td>
			<td>100499-578</td>
			<td></td>
		</tr>
		<tr>
			<td>Feather Scalpel (No. 11, 40 mm)</td>
			<td>VWT</td>
			<td>100499-580</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl (99%)</td>
			<td>Sigma</td>
			<td>P-4504</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimble Kontes pellet pestle</td>
			<td>Sigma</td>
			<td>z359971</td>
			<td></td>
		</tr>
		<tr>
			<td>Labconco Fast-Freeze Flasks</td>
			<td>Labconco</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>LN2 (liquid nitrogen) + Dewar flask or similar vacuum flask</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>M-9272</td>
			<td></td>
		</tr>
		<tr>
			<td>Milli-Q/de-ionized water</td>
			<td>EMD Millipore</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4 (powder)</td>
			<td>J.T. Baker</td>
			<td>4062-01</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl (crystal)</td>
			<td>EMD Millipore</td>
			<td>Sx0420-3</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Amresco</td>
			<td>0865</td>
			<td></td>
		</tr>
		<tr>
			<td>OmniPur EDTA</td>
			<td>EMD</td>
			<td>4005</td>
			<td></td>
		</tr>
		<tr>
			<td>OmniPur HEPES, Free Acid</td>
			<td>EMD</td>
			<td>5320</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm M</td>
			<td>Sigma-Aldrich</td>
			<td>P7793</td>
			<td></td>
		</tr>
		<tr>
			<td>Reynolds Wrap Aluminum Foil</td>
			<td>Reynolds Brands</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Scotch Magic Tape (12.7 mm x 32.9 m)</td>
			<td>Scotch-3M</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium deoxycholate</td>
			<td>Sigma-Aldrich</td>
			<td>D67501</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrafuge mini centrifuge</td>
			<td>Labnet International, Inc</td>
			<td>C1301</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue incubation chamber (purchased or custom made)</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>J.T.Baker</td>
			<td>4103-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Signma-Aldrich</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>VirTis Benchtop 2K Lyophilizer or equivalent machine</td>
			<td>SP Scientific</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>VWR Grade 413 Filer Paper (diameter 5.5 cm, pore size 5 &#956;m)</td>
			<td>VWR</td>
			<td>28310-015</td>
			<td></td>
		</tr>
		<tr>
			<td>Whatman Grade 1 Qualitative Filter Paper (diameter 9 cm, pore size 11 &#956;m)</td>
			<td>Whatman/GE Healthcare</td>
			<td>1001-090</td>
			<td></td>
		</tr>
		<tr>
			<td>Wide bore transfer pipet, Global Scientific</td>
			<td>VWR</td>
			<td>76285-362</td>
			<td></td>
		</tr>
	</tbody>
</table>

two peeling methods for the isolation of photoreceptor cell compartments in the mouse retina for protein analysis

Rod photoreceptors are highly polarized sensory neurons with distinct compartments. Mouse rods are long (~80 &#181;m) and thin (~2 &#181;m) and are laterally packed in the outermost layer of the retina, the photoreceptor layer, resulting in alignment of analogous subcellular compartments. Traditionally, tangential sectioning of the frozen flat-mounted retina has been used to study the movement and localization of proteins within different rod compartments. However, the high curvature of the rod-dominant mouse retina makes tangential sectioning challenging. Motivated by the study of protein transport between compartments, we developed two peeling methods that reliably isolate the rod outer segment (ROS) and other subcellular compartments for western blots. Our relatively quick and simple techniques deliver enriched and subcellular-specific fractions to quantitatively measure the distribution and redistribution of important photoreceptor proteins in normal rods. Moreover, these isolation techniques can also be easily adapted to isolate and quantitatively investigate the protein composition of other cellular layers within both healthy and degenerating retinae.

Watch this Scientific Journal Video about Two Peeling Methods for the Isolation of Photoreceptor Cell Compartments in the Mouse Retina for Protein Analysis at JoVE.com

Two Peeling Methods for the Isolation of Photoreceptor Cell Compartments in the Mouse Retina for Protein Analysis

This protocol presents two techniques to isolate the subcellular compartments of murine rod photoreceptors for protein analysis. The first method utilizes live retinae and cellulose filter paper to separate rod outer segments, while the second employs lyophilized retinae and adhesive tape to peel away rod inner and outer segment layers.

Rod photoreceptors are highly polarized sensory neurons with distinct compartments. Mouse rods are long (~80 &#181;m) and thin (~2 &#181;m) and are ...

two-peeling-methods-for-isolation-photoreceptor-cell-compartments

Nanyang Technological University

Research

JoVE Journal

Neuroscience

3.0K Views.  University of Southern California. This protocol presents two techniques to isolate the subcellular compartments of murine rod photoreceptors for protein analysis. The first method utilizes live retinae and cellulose filter paper to separate rod outer segments, while the second employs lyophilized retinae and adhesive tape to peel away rod inner and outer segment layers.

Video: Two Peeling Methods for the Isolation of Photoreceptor Cell Compartments in the Mouse Retina for Protein Analysis

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

Methods for the Modulation and Analysis of NF-&#954;B-dependent Adult Neurogenesis

The hippocampus plays a pivotal role in the formation and consolidation of episodic memories, and in spatial orientation. Historically, the adult hippocampus has been viewed as a very static anatomical region of the mammalian brain. However, recent findings have demonstrated that the dentate gyrus of the hippocampus is an area of tremendous plasticity in adults, involving not only modifications of existing neuronal circuits, but also adult neurogenesis. This plasticity is regulated by complex transcriptional networks, in which the transcription factor NF-&#954;B plays a prominent role. To study and manipulate adult neurogenesis, a transgenic mouse model for forebrain-specific neuronal inhibition of NF-&#954;B activity can be used.
In this study, methods are described for the analysis of NF-&#954;B-dependent neurogenesis, including its structural aspects, neuronal apoptosis and progenitor proliferation, and cognitive significance, which was specifically assessed via a dentate gyrus (DG)-dependent behavioral test, the spatial pattern separation-Barnes maze (SPS-BM). The SPS-BM protocol could be simply adapted for use with other transgenic animal models designed to assess the influence of particular genes on adult hippocampal neurogenesis. Furthermore, SPS-BM could be used in other experimental settings aimed at investigating and manipulating DG-dependent learning, for example, using pharmacological agents.

Methods for the manipulation and analysis of NF-&#954;B-dependent adult hippocampal neurogenesis are described. A detailed protocol is presented for a dentate gyrus-dependent behavioral test (termed the spatial pattern separation-Barnes maze) for the investigation of cognitive outcome in mice. This technique should also help enable investigations in other experimental settings.

The hippocampus plays a pivotal role in the formation and consolidation of episodic memories, and in spatial orientation. Historically, the adult ...

One Mouse, Two Cultures: Isolation and Culture of Adult Neural Stem Cells from the Two Neurogenic Zones of Individual Mice

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult neural stem cells in vitro. These assays can be used to compare the precursor potential of cells isolated from genetically different or differentially treated animals&#160;to determine the effects of exogenous factors on neural precursor cell proliferation and differentiation and to generate neural precursor cell lines that can be assayed over continuous passages. The neurosphere assay is traditionally used for the post-hoc identification of stem cells, primarily due to the lack of definitive markers with which they can be isolated from primary tissue&#160;and has the major advantage of giving a quick estimate of precursor cell numbers in brain tissue derived from individual animals. Adherent monolayer cultures, in contrast, are not traditionally used to compare proliferation between individual animals, as each culture is generally initiated from the combined tissue of between 5-8 animals. However, they have the major advantage that, unlike neurospheres, they consist of a mostly homogeneous population of precursor cells and are useful for following the differentiation process in single cells. Here, we describe, in detail, the generation of neurosphere cultures and, for the first time, adherent cultures from individual animals. This has many important implications including paired analysis of proliferation and/or differentiation potential in both the subventricular zone (SVZ) and dentate gyrus (DG) of treated or genetically different mouse lines, as well as a significant reduction in animal usage.

Here we describe a detailed protocol for the simultaneous generation of neural precursor cell cultures, as either adherent monolayers or neurospheres, from the subventricular zone and dentate gyrus of individual adult mice.

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult ...

Assessment of Vascular Regeneration in the CNS Using the Mouse Retina

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). It is increasingly being recognized that several neurodegenerative diseases such as Alzheimer&#8217;s, multiple sclerosis, and amyotrophic lateral sclerosis present elements of vascular compromise. In addition, the most prominent causes of blindness in pediatric and working age populations (retinopathy of prematurity and diabetic retinopathy, respectively) are characterized by vascular degeneration and failure of physiological vascular regrowth. The aim of this technical paper is to provide a detailed protocol to study CNS vascular regeneration in the retina. The method can be employed to elucidate molecular mechanisms that lead to failure of vascular growth after ischemic injury. In addition, potential therapeutic modalities to accelerate and restore healthy vascular plexuses can be explored. Findings obtained using the described approach may provide therapeutic avenues for ischemic retinopathies such as that of diabetes or prematurity and possibly benefit other vascular disorders of the CNS.

The rodent retina has long been recognized as an accessible window to the brain. In this technical paper we provide a protocol that employs the mouse model of oxygen-induced retinopathy to study the mechanisms that lead to failure of vascular regeneration within the central nervous system after ischemic injury. The described system can also be harnessed to explore strategies to promote regrowth of functional blood vessels within the retina and CNS.

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). ...

Vibratome Sectioning Mouse Retina to Prepare Photoreceptor Cultures

The retina is a part of the central nervous system that has organized architecture, with neurons in layers from the photoreceptors, both rods and cones in contact with the retinal pigmented epithelium in the most distant part on the retina considering the direction of light, and the ganglion cells in the most proximal distance. This architecture allows the isolation of the photoreceptor layer by vibratome sectioning. The dissected neural retina of a mouse aged 8 days is flat-embedded in 4% gelatin on top of a slice of 20% gelatin photoreceptor layer facing down. Using a vibratome and a double edged razor blade, the 100 &#181;m thick inner retina is sectioned. This section contains the ganglion cells and the inner layer with notably the bipolar cells. An intermediary section of 15 &#181;m is discarded before 200 &#181;m of the outer retina containing the photoreceptors is recovered. The gelatin is removed by heating at 37 &#176;C. Pieces of outer layer are incubated in 500 &#181;l of Ringer&#39;s solution with 2 units of activated papain for 20 min at 37 &#176;C. The reaction is stopped by adding 500 &#181;l 10% fetal calf serum (FCS) in Dulbecco&#39;s Modified Eagle Medium (DMEM), then 25 units of DNAse I is added before centrifugation at RT, washed several times to remove serum and the cells are resuspended in 500 &#181;l of DMEM and seeded at 1 x 105 cells/cm2. The cells are grown to 5 days in vitro and their viability scored using live/dead assay. The purity of the culture is first determined by microscopic observation during the experiment. The purity is then validated by seeding and fixing cells on a histological slide and analyzing using a rabbit polyclonal anti-SAG, a photoreceptor marker and mouse monoclonal anti-RHO, a rod photoreceptor specific marker. Alternatively, the photoreceptor layer (97% rods) can be used for gene or protein expression analysis and for transplantation.

Neural retina of a mouse aged 8 days is on top of a 4% gelatin block. After isolation of the photoreceptor layer (200 &#181;m) by vibratome, the photoreceptors are seeded after mechanical and enzymatic dissociation for culture. The photoreceptor layer can be used for molecular, biochemical analyses or transplantation.

The retina is a part of the central nervous system that has organized architecture, with neurons in layers from the photoreceptors, both rods and cones in ...

Methods for Cell-attached Capacitance Measurements in Mouse Adrenal Chromaffin Cell

Neuronal transmission is an integral part of cellular communication within the brain. Depolarization of the presynaptic membrane leads to vesicle fusion known as exocytosis that mediates synaptic transmission. Subsequent retrieval of synaptic vesicles is necessary to generate new neurotransmitter-filled vesicles in a process identified as endocytosis. During exocytosis, fusing vesicle membranes will result in an increase in surface area and subsequent endocytosis results in a decrease in the surface area. Here, our lab demonstrates a basic introduction to cell-attached capacitance recordings of single endocytic events in the mouse adrenal chromaffin cell. This type of electrical recording is useful for high-resolution recordings of exocytosis and endocytosis at the single vesicle level. While this technique can detect both vesicle exocytosis and endocytosis, the focus of our lab is vesicle endocytosis. Moreover, this technique allows us to analyze the kinetics of single endocytic events. Here the methods for mouse adrenal gland tissue dissection, chromaffin cell culture, basic cell-attached techniques, and subsequent examples of individual traces measuring singular endocytic event are described.

After exocytosis, fused plasma membrane is retrieved through a process known as endocytosis. This mechanism reforms new synaptic vesicles for the next round of release. Individual endocytic events are captured and analyzed through the use of the cell-attached capacitance recordings in mouse adrenal chromaffin cells.

Neuronal transmission is an integral part of cellular communication within the brain. Depolarization of the presynaptic membrane leads to vesicle fusion ...

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to blindness in many retinal diseases. Calcium (Ca2+), a key second messenger in photoreceptor signaling and metabolism, has been proposed to be indirectly linked with photoreceptor degeneration in various animal models. Systematically studying these aspects of cone physiology and pathophysiology has been hampered by the difficulties of electrically recording from these small cells, in particular in the mouse where the retina is dominated by rod photoreceptors. To circumvent this issue, we established a two-photon Ca2+ imaging protocol using a transgenic mouse line that expresses the genetically encoded Ca2+ biosensor TN-XL exclusively in cones and can be crossbred with mouse models for photoreceptor degeneration. The protocol described here involves preparing vertical sections (&#8220;slices&#8221;) of retinas from mice and optical imaging of light stimulus-evoked changes in cone Ca2+ level. The protocol also allows &#8220;in-slice measurement&#8221; of absolute Ca2+ concentrations; as the recordings can be followed by calibration. This protocol enables studies into functional cone properties and is expected to contribute to the understanding of cone Ca2+ signaling as well as the potential involvement of Ca2+ in photoreceptor death and retinal degeneration.

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

We describe a protocol to monitor Ca2+ dynamics in the axon terminals of cone photoreceptors using an ex-vivo&#160;slice preparation of the mouse retina. This protocol allows comprehensive studies of cone Ca2+ signaling in an important mammalian model system, the mouse.

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to ...

In-depth Physiological Analysis of Defined Cell Populations in Acute Tissue Slices of the Mouse Vomeronasal Organ

In most mammals, the vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Vomeronasal sensory neurons (VSNs) express a specific type of G protein-coupled receptor (GPCR) from at least three different chemoreceptor gene families allowing sensitive and specific detection of chemosensory cues. These families comprise the V1r and V2r gene families as well as the formyl peptide receptor (FPR)-related sequence (Fpr-rs) family of putative chemoreceptor genes. In order to understand the physiology of vomeronasal receptor-ligand interactions and downstream signaling, it is essential to identify the biophysical properties inherent to each specific class of VSNs.
The physiological approach described here allows identification and in-depth analysis of a defined population of sensory neurons using a transgenic mouse line (Fpr-rs3-i-Venus). The use of this protocol, however, is not restricted to this specific line and thus can easily be extended to other genetically modified lines or wild type animals.

Here, we describe a physiological approach that allows identification and in-depth analysis of a defined population of sensory neurons in acute coronal tissue slices of the mouse vomeronasal organ using whole-cell patch-clamp recordings.

In most mammals, the vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Vomeronasal sensory ...

Where You Cut Matters: A Dissection and Analysis Guide for the Spatial Orientation of the Mouse Retina from Ocular Landmarks

Accurately and reliably identifying spatial orientation of the isolated mouse retina is important for many studies in visual neuroscience, including the analysis of density and size gradients of retinal cell types, the direction tuning of direction-selective ganglion cells, and the examination of topographic degeneration patterns in some retinal diseases. However, there are many different ocular dissection methods reported in the literature that are used to identify and label retinal orientation in the mouse retina. While the method of orientation used in such studies is often overlooked, not reporting how retinal orientation is determined can cause discrepancies in the literature and confusion when attempting to compare data between studies. Superficial ocular landmarks such as corneal burns are commonly used but have recently been shown to be less reliable than deeper landmarks such as the rectus muscles, the choroid fissure, or the s-opsin gradient. Here, we provide a comprehensive guide for the use of deep ocular landmarks to accurately dissect and document the spatial orientation of an isolated mouse retina. We have also compared the effectiveness of two s-opsin antibodies and included a protocol for s-opsin immunohistochemistry. Because orientation of the retina according to the s-opsin gradient requires retinal reconstruction with Retistruct software and rotation with custom code, we have presented the important steps required to use both of these programs. Overall, the goal of this protocol is to deliver a reliable and repeatable set of methods for accurate retinal orientation that is adaptable to most experimental protocols. An overarching goal of this work is to standardize retinal orientation methods for future studies.

This protocol provides a comprehensive dissection and analysis guide for the use of deep ocular landmarks, s-opsin immunohistochemistry, Retistruct, and custom code to accurately and reliably orient the isolated mouse retina in anatomical space.

Accurately and reliably identifying spatial orientation of the isolated mouse retina is important for many studies in visual neuroscience, including the ...

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging methods exist for examining the brain tissue of live newborn mammals. Herein we summarize a protocol for imaging individual cortical neurons in living neonatal mice. This protocol includes the following two methodologies: (1) the Supernova system for sparse and bright labeling of cortical neurons in the developing brain, and (2) a surgical procedure for the fragile neonatal skull. This protocol allows the observation of temporal changes of individual cortical neurites during neonatal stages with a high signal-to-noise ratio. Labeled cell-specific gene silencing and knockout can also be achieved by combining the Supernova with RNA interference and CRISPR/Cas9 gene editing systems. This protocol can, thus, be used for analyzing the developmental dynamics of cortical neurons, molecular mechanisms that control the neuronal dynamics, and changes in neuronal dynamics in disease models.

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

We present an in vivo two-photon imaging protocol for imaging the cerebral cortex of neonatal mice. This method is suitable for analyzing the developmental dynamics of cortical neurons, the molecular mechanisms that control the neuronal dynamics, and the changes in neuronal dynamics in disease models.

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging ...

Two Peeling Methods for the Isolation of Photoreceptor Cell Compartments in the Mouse Retina for Protein Analysis

Podsumowanie

Przeglądaj więcej filmów

Rozdziały w tym wideo

In vivo Electroporation of Developing Mouse Retina

Methods for the Modulation and Analysis of NF-κB-dependent Adult Neurogenesis

One Mouse, Two Cultures: Isolation and Culture of Adult Neural Stem Cells from the Two Neurogenic Zones of Individual Mice

Assessment of Vascular Regeneration in the CNS Using the Mouse Retina

Vibratome Sectioning Mouse Retina to Prepare Photoreceptor Cultures

Methods for Cell-attached Capacitance Measurements in Mouse Adrenal Chromaffin Cell

Imaging Ca²⁺ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

In-depth Physiological Analysis of Defined Cell Populations in Acute Tissue Slices of the Mouse Vomeronasal Organ

Where You Cut Matters: A Dissection and Analysis Guide for the Spatial Orientation of the Mouse Retina from Ocular Landmarks

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice