Igal Gery and David Hicks for anti-SAG antibodies. Ram Fridlich for reading the manuscript.

NOTE: The procedure was approved by the ethics committee Darwin from University Pierre and Marie Curie (Ce5/2009/048)
1. Preparation of Gelatin Solution, Instruments, Vibratome, Culture Media and Culture Plate
<ol>
	<li>Prepare a 20% gelatin solution at least one day before the experiment.
	<ol>
		<li>Under the hood, add 500 &#181;l of gentamycin [10 mg/ml] to a 500 ml bottle containing CO2 independent medium (CO2-i).</li>
		<li>In a separate beaker, add 20 ml of CO2-I (from step 1.1.1) and heat at 95 &#176;C on a heating block with constant stirring for 15 min. Observe a color change to yellow, indicating that the solution is ready for further use.</li>
		<li>Weigh 4 g of gelatin and gradually poor to the solution prepared above (step 1.1.2) with increased stirring and heating at 95 &#176;C for 45 min, to obtain a yellow color solution. Allow the gelatin solution to cool under the hood for approximately 5 min.</li>
		<li>Pour 4 ml of the gelatin solution into a 35 mm diameter culture dishes without introducing air bubbles. Keep the dish at 21 &#176;C for 30 min. Cover the dishes with plastic film, and turn the dishes upside down. Optionally, store the dishes at 4 &#176;C for up to 2 months before use.</li>
	</ol>
	</li>
	<li>Prepare a 4% gelatin solution.
	<ol>
		<li>Under the hood, add 25 ml of CO2-i medium in a sterile plastic container, and heat at 42 &#176;C on a heating block. Remove the medium from the heating block and gradually add 1 g of gelatin to the hot medium and stir. Quickly return the container on the heating block (42 &#176;C) and keep it warm during the process.</li>
	</ol>
	</li>
	<li>Setting-up the vibratome apparatus:
	<ol>
		<li>Remove the dishes containing 20% gelatin solution stored at 4 &#176;C. Cut the gelatin with a scalpel. Flip the gelatin slice and stick it on the black support disc of the vibratome using a drop of super glue.</li>
		<li>Break a razor blade into two halves, and insert one half into the vibratome holding receptacle. Insert the black support disc onto the vibratome apparatus. Turn on the black knob to the right side. Fix the holding receptacle on the head of the vibratome. Add sufficient amount of CO2-i medium in the vibratome tank to cover the gelatin block.</li>
		<li>Switch on the vibratome and cut three 100 &#181;m slices of gelatin block. Keep the gelatin block for further use.</li>
	</ol>
	</li>
	<li>Prepare 40 ml of culture medium:
	<ol>
		<li>Prepare Dulbecco&#39;s modified Eagle medium (DMEM) with 10% of fetal calf serum (FCS) under the hood. Prepare 1 mg/ml Poly-D-Lysine using phosphate buffer saline (PBS) pH 7.4.</li>
		<li>In a 96-well plate, add 2 &#181;g/cm2 of Poly-D-Lysine to coat the bottom of the wells. Incubate the plate for 45 min at 37 &#176;C within 5% CO2 incubator. Following incubation, remove PBS and replace with 200 &#181;l / well DMEM and place again at 37 &#176;C in 5% CO2 incubator before using.</li>
	</ol>
	</li>
</ol>
2. Dissection of Entire Retina
<ol>
	<li>Enucleate the mouse:
	<ol>
		<li>Sacrifice the mouse according to the European directive 2010/63: by cervical dislocation. Cut the head with curved-scissors and place it in a 100 mm diameter Petri dish after having disinfected the eye with disinfectant.</li>
		<li>Remove the eye after having detached the optic nerve by sectioning it very carefully using a curved grip. Then place both eyes in a 35 mm Petri dish containing sterile CO2-i medium at 21 &#176;C.</li>
	</ol>
	</li>
	<li>Removing the retina:
	<ol>
		<li>Make a hole at the level of the eye-limb with the help of a 18 G needle to be able to introduce the scissors into the eye globe. Introduce straight scissors inside the hole and carefully cut the sclera below the iris on all the perimeter of the ocular globe to remove ciliary body.</li>
		<li>Remove the cornea and the lens and keep the posterior chamber of the eye with the retina contiguous to the sclera. With two fine grips, remove the vitreous located inside the posterior chamber without damaging the retina. Perform four radial cuts (retina/sclera) to permit flattening of the retina.</li>
		<li>Upturn the ocular globe and peel it as an &#8220;orange&#8221;. Detach the retina from the sclera and retinal pigmented epithelium (RPE). Detach the retina still attached at the level of the optic nerve very carefully with fine curved-scissors.</li>
		<li>Detach the remaining vitreous from the periphery toward the centre of the retina with two fine grips. Make sure that all the vitreous is completely removed to allow the flattening of the retina.</li>
		<li>Cut the extremity of a plastic pipette and transfer the retina to a 35 mm diameter Petri dish containing CO2-i medium with the plastic pipette.</li>
	</ol>
	</li>
</ol>
3. Sealing of Flat-mounted Retina onto Gelatin Block
<ol>
	<li>Remove 20 - 30 ml of CO2-i medium from the vibratome tank (the gelatin block is free) to permit the sealing of flat-mounted retina.</li>
	<li>Transfer the retina to a glass slide using a plastic pipette and add a drop of CO2-i medium before transferring to the gelatin slice with the photoreceptor facing down on the gelatin slice.</li>
	<li>Attach the retina to the gelatin block by gently injecting warmed 4% gelatin on one side of the block between the retina and the gelatin block and simultaneously expelling warm gelatin on the other side.</li>
	<li>Aspirate 4% gelatin with a pasteur pipette. Wait for 10 min to allow total sealing. Add CO2-i medium to immerse the block and the blade completely.</li>
</ol>
4. Sectioning the Retina

NOTE: This step is critical to obtain a layer of photoreceptor cells without the other retinal cells (Figure 1).
<ol>
	<li>Starting from the top on the gelatin block, cut 100 &#181;m serial sections to reach the retina. Then cut 100 - 120 &#181;m sections of the inner layer. Depending on the application, this layer may be discarded or stored in liquid nitrogen. Ensure that the speed of the vibratome is very slow (at 1 or 2) during the sectioning of the inner layer or photoreceptors layers.</li>
	<li>Perform intermediate 15 &#181;m sections of the retina corresponding to the interface between the inner and the outer retina. Observe this section under a microscope. The absence of blood vessels indicates that the outer retina has been reached.</li>
	<li>Cut 200 &#181;m of the outer retina (the photoreceptor layer) with gelatin and transfer it to a 35 mm diameter dish filled with 3 ml of CO2-i medium. Keep it on ice until all the photoreceptor layer sections of all retinas are obtained.</li>
</ol>
5. Dissociating Photoreceptor Cells
<ol>
	<li>Take the dish(es) containing the photoreceptor layer(s) and put it(them) in an incubator at 37 &#176;C for 10 min.</li>
	<li>Using forceps gently separate the photoreceptor layer from the gelatin and transfer it in a new dish filled with 3 ml of Ringer&#8217;s solution. Repeat this step (5.2) for each photoreceptor layer preparation.</li>
	<li>Incubate 2 units of papain with 25 &#181;l of activator solution (1.1 mM EDTA; 5.5 mM L-cystein; 60 &#181;M &#946;-mercaptoethanol) in a 5 ml polypropylene sterile tube and incubate it 30 min at 37 &#176;C within 5% CO2 incubator. The activation of papain is achieved at 37 &#176;C. During this incubation, cut the photoreceptor layer into 2 mm2 pieces (not too small) and transfer these pieces into a 5 ml tubes.</li>
	<li>Rinse the retina twice with 1.5 ml of solution Ringer&#8217;s solution followed by gravity sedimentation. Remove all remaining Ringer&#8217;s solution from the tube with the specimen.</li>
	<li>Add 475 &#181;l of Ringer&#8217;s solution to the tube containing activated papain and mix. Add this solution to the tube containing the retina.</li>
	<li>Incubate the tube with the retina for 20 min at 37&#176;C within 5% CO2 incubator. Stop the reaction by adding 1 ml of 10% FCS in DMEM. Add 25 U of deoxyribonuclease I (DNAse I) to digested DNA from death cells. Carefully homogenize the cell suspension using a 1 ml-pipette. Spin at 50 x g for 6 min at RT.</li>
	<li>Discard the supernatant to remove traces of serum and add DMEM medium with supplements (see table of specific reagents_equipment) to the cell-pellet and resuspend carefully the cell suspension with a 1 ml pipette. Spin at 50 x g for 6 min at RT. Repeat this step (5.7) once more.</li>
</ol>
6 Culturing Photoreceptor Cells
<ol>
	<li>The photoreceptor layer of a mouse at PN8 contains about 1 to 1.5 million cells using this technique. Seed the photoreceptor cells at 1 x 105 cells/cm2 in a culture plate with culture medium and culture the cells for 5 days at 37 &#176;C within 5% CO2 incubator. On day 5, proceed to immunochemistry and western blotting study following methods provided by the antibody suppliers. 
	NOTE: Steps to stop the culture and preliminary tests are described in reference 21.</li>
</ol>

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RetNet, the Retinal Information Network Available from: <a target="_blank" href="https://sph.uth.edu/retnet">https://sph.uth.edu/retnet</a> (2014)</li><li>Mohand-Said, S., Hicks, D., Dreyfus, H., Sahel, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+transplantation+of+rods+delays+cone+loss+in+a+retinitis+pigmentosa+model.">Selective transplantation of rods delays cone loss in a retinitis pigmentosa model.</a> Arch Ophthalmol. 118 (6), 807-811 (2000).</li><li>MacLaren, R. 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=Retinal+repair+by+transplantation+of+photoreceptor+precursors.">Retinal repair by transplantation of photoreceptor precursors.</a> Nature. 444 (7116), 203-207 (2006).</li><li>Yang, Y., 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=Transplantation+of+photoreceptor+and+total+neural+retina+preserves+cone+function+in+P23H+rhodopsin+transgenic+rat.">Transplantation of photoreceptor and total neural retina preserves cone function in P23H rhodopsin transgenic rat.</a> PLoS One. 5 (10), (2010).</li><li>Mohand-Said, 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=Normal+retina+releases+a+diffusible+factor+stimulating+cone+survival+in+the+retinal+degeneration+mouse.">Normal retina releases a diffusible factor stimulating cone survival in the retinal degeneration mouse.</a> Proc Natl Acad Sci USA. 95 (14), 8357-8362 (1998).</li><li>Fintz, A. 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H., Holmgren, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thioredoxin+and+related+molecules--from+biology+to+health+and+disease.">Thioredoxin and related molecules--from biology to health and disease.</a> Antioxid Redox Signal. 9 (1), 25-47 (2007).</li><li>Fridlich, 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=The+thioredoxin-like+protein+rod-derived+cone+viability+factor+(RdCVFL)+interacts+with+TAU+and+inhibits+its+phosphorylation+in+the+retina.">The thioredoxin-like protein rod-derived cone viability factor (RdCVFL) interacts with TAU and inhibits its phosphorylation in the retina.</a> Mol Cell Proteomics. 8 (6), 1206-1218 (2009).</li><li>Yang, Y., 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=Functional+cone+rescue+by+RdCVF+protein+in+a+dominant+model+of+retinitis+pigmentosa.">Functional cone rescue by RdCVF protein in a dominant model of retinitis pigmentosa.</a> Mol Ther. 17 (5), 787-795 (2009).</li><li>Bennett, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+for+delivery+of+rod-derived+cone+viability+factor.">Strategies for delivery of rod-derived cone viability factor.</a> Retina. 25 (8 Suppl), S47 (2005).</li><li>Komaromy, A. 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The authors have nothing to disclose.

The retina is a model organ in biology. Study of the retina led to 6 major discoveries in biology. It is at the origin of the first tumor suppressor gene RB1. It reveals the molecular link between the receptor tyrosine kinases and the MAP kinases through the interaction with Son of sevenless. It was involved in the discovery of PAX6, the first master control gene for organ morphogenesis. It is at the center of the genetic association of Complement factor H (CFH) with age-related macular degeneration (AMD), the first disease susceptibility gene identified by genome wide association screen (GWAS). Finally, it led to the first successful gene therapy for Leber congenital amaurosis, the first corrective gene therapy trial in human of any inherited disease. The structure of this organ is conserved in most vertebrate species. Its accessibility for manipulation in vivo has encouraged early on functional genomics investigations on this integral part of the central nervous system.
We show here how to separate the photoreceptor layer from the inner layer of the retina by vibratome sectioning. This step is critical to obtain pure photoreceptor cultures. Our dissection protocol makes the contamination by RPE cells very unlikely.
One of the major challenges is the flattening of the retina that is necessary to section properly the inner and the outer retina. The sectioning of the retina is best on retina with small diameter as those from rodents and this diameter is a limitation of the technique with current available material.
It is advisable before starting a biologically meaningful experiment, to practice on a sample of the retina. We illustrate the method by representative results obtained from cultured photoreceptor cells, using the material to perform expression study of both mRNAs and proteins. Expression studies can also be performed alternatively on sections obtained by laser capture microdissection, but the cultures are best performed using vibratome sectioning. We could have used the technique of microdissection laser with a complete different strategy. But, to collect the photoreceptor layer with the microdissection apparatus for culture, it would be necessary to avoid fixative and this would complicate the current methodology significantly.
We are developing a protocol aimed at studying the kinetics of gene and protein expression in vibratome sections during the degeneration of photoreceptors in models of retinitis pigmentosa. We believe that the detailed description of the protocol will be useful to researchers in the field of retinal biology, and most particularly for proteomic and metabolomic studies.

The retina is an integral part of the central nervous system which has a conserved architecture among vertebrates. The neurons of the neural retina are organized in layers, with the most distant for the incident light, the photoreceptor layer in close contact with the retinal pigmented epithelium (RPE) at the back of the eye. Rod and cone photorecptors are light sensitive cells that rely on opsin sensitive molecules for photon capture. These molecules are enclosed on disk membranes of a cellular structure, located on the outer segment of the photoreceptor which points in the direction of the RPE1. This structure, which is most often affected very early in cases of photoreceptor degenerations, is renewed at the rate of 10% every day. The so called inner layer contains most of the other neurons that compute the signal received from the photoreceptors, the bipolar, amacrine and horizontal cells as well as the ganglion cells. These latter with their axons form a beam which is the optic nerve. This layering is so conserved that biologists have used the term displaced amacrine cells when the cells are found outside the inner plexiform layer2. Layers of neurons are distributed within an armature of radial M&#252;ller glial cells. Bipolar cells link photoreceptors to ganglion cells. They are located between the external plexiform layer and the inner plexiform layer. The ganglion cells form the inner plexiform layer in connection with the bipolar cells. The amacrin cells are named as association cells located in the inner plexiform layer between the bipolar cells and ganglion cells. The outer plexiform layer contains horizontal cells. This unique arrangement of neuronal layers of the central nervous system permits the isolation of the photoreceptor layer from the inner cell layer by slicing the flat-mounted retina using a vibratome.
Originally, this technique was used to isolate photoreceptors for transplantation in the eye of the rd1 mouse, a model of human retinitis pigmentosa (RP)3. The rd1 mouse carries a recessive mutation in the Pde6b gene which encodes for the rod-specific phosphodiesterase beta subunit. Recessive mutations of this gene result in RP in humans4. After rod photoreceptors have degenerated, the patient loses night vision, and surprisingly cone photoreceptors, which do not express the mutated gene, degenerate as a second step. Because the cones are required for color vision and visual acuity, the patients become progressively blind and an effective treatment for the disease has not yet developed. By grafting photoreceptor layer from a wild-type mouse the cone degeneration of the host mouse is delayed3,5. The rods lost in the rod-cone degenerative model could not be replaced by a transplant because the synaptic connection between rods and bipolar cells can only be obtained at a specific stage of retinal development, marked by the onset of Nrl expression6. The layer of photoreceptors is introduced by surgery in the sub-retinal space of the rod-less rd1 retina, between the RPE and the outer retina corresponding to only 3% of the remaining photoreceptors, the cones. Two weeks after surgery, 40% of the cones from the animal transplanted with a normal photoreceptor layer survived as compared to the animal transplanted with a normal layer of inner retinal cells, or to the sham-operated animal. The topography of cone survival, spread-out over the entire surface of the mutated retina located at the position of the grafted tissue indicates that the protective effect is due to a diffusible molecule7.
Next, we used a co-culture system as well as conditioned culture media to validate the fact that the protective effect relies on the secretion of a protein by rods8,9. We hypothesize that this protein will be expressed continuously and specifically by rods and that their death during the first phase of the disease will trigger secondary cone degeneration by the loss of a protective signal from rods in a non-cell autonomous manner10. Because of the importance of cone mediated central vision in primates this putative protein will be a highly relevant therapeutic tool for RP. Preserving the cones in RP would theoretically prevent a total of 1.5 million patients worldwide to become blind11. We have used a high content screening approach and a cone-enriched culture model to identify a cDNA encoding Rod-derived Cone Viability Factor (RdCVF) from a retinal cDNA library12. RdCVF is the spliced product of the NXNL1 gene which, interestingly is homologous to the gene encoding for thioredoxin proteins involved in redox homeostasis13. The second spliced product of the gene, RdCVFL is an enzyme that protects its target, the TAU protein against oxidative damage14. Administration of RdCVF prevents the secondary degeneration of cones and the loss of their visual function in a recessive, and dominant model of RP12,15. This demonstrates two important aspects of this innovative therapeutic strategy16. First, it can be applied in most of the RP cases in a gene-independent manner. Second, contrary to the competing factor CNTF, RdCVF survival is associated with the maintenance of visual function17. The absence of functional effect may explain the reason of the absence of clinical benefit of the administration of CNTF to RP patients18. RdCVF is most likely one of the important survival signal between rods and cones since cone rescue in vitro is inhibited by RdCVF immunodepletion12. In addition, the disruption of the rod-derived cone viability gene leads to photoreceptor dysfunction and susceptibility to oxidative stress19.
The use of photoreceptor layer is at the origin of identification of RdCVF and of a novel redox signal involved in neurodegenerative diseases20. This manuscript describes the protocol used to isolate and culture cells from the photoreceptor layer to characterize the activity of RdCVF. The photoreceptors can be maintained in culture for 5 to 7 days21. This technique can also be used to study the expression of photoreceptor specific genes.

<table><tbody><tr><td>Cytidine 5&amp;prime;-diphosphocholin*</td><td>Sigma-Aldrich</td><td>C0256</td><td>4.7 &amp;micro;M Cytidine 5&amp;prime;-diphosphocholin</td></tr><tr><td>Cytidine 5&amp;prime;-diphosphoethanolamine*</td><td>Sigma-Aldrich</td><td>C0456</td><td>2.7 &amp;micro;M Cytidine 5&amp;prime;-diphosphoethanolamine</td></tr><tr><td>Linole&amp;iuml;c acid/ bovine serum albumin (BSA)*</td><td>Sigma-Aldrich</td><td>L8384</td><td>100 &amp;micro;g/ml linole&amp;iuml;c acid/ bovine serum albumin (BSA)</td></tr><tr><td>Triiodo-L-thyronine*</td><td>Sigma-Aldrich</td><td>T6397</td><td>0.03 &amp;micro;M Triiodo-L-thyronine</td></tr><tr><td>96-well plates</td><td>Greiner bio-one</td><td>655-095</td><td></td></tr><tr><td>Binocular microscope</td><td>Leica</td><td>MZ-75</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; independent (CO&lt;sub&gt;2-&lt;/sub&gt;i)</td><td>Life Technologies</td><td>18045054</td><td></td></tr><tr><td>DMEM</td><td>Life Technologies</td><td>41966029</td><td></td></tr><tr><td>Forceps n&amp;deg;5 Dumont</td><td>Bionic</td><td>11254-20</td><td></td></tr><tr><td>Gelatin from porcin skin type A</td><td>Sigma-Aldrich</td><td>G2500</td><td></td></tr><tr><td>GeneChip&amp;nbsp;</td><td>Affymetrix</td><td>U74v2</td><td></td></tr><tr><td>Gentamicin solution</td><td>Life Technologies</td><td>15710049</td><td></td></tr><tr><td>Hydrocortison*</td><td>Sigma-Aldrich</td><td>H0888</td><td>0.55 &amp;micro;M hydrocortison</td></tr><tr><td>Insulin* (I)</td><td>Sigma-Aldrich</td><td>I1884 (ITS)</td><td>0.86 &amp;micro;M insulin (I)</td></tr><tr><td>Papain</td><td>Worthington-biochem</td><td>WOLS03124</td><td></td></tr><tr><td>Poly-D-lysine</td><td>Sigma-Aldrich</td><td>P-6407</td><td></td></tr><tr><td>Progesterone*</td><td>Sigma-Aldrich</td><td>P7556&amp;nbsp;</td><td>2.0 &amp;micro;M progesterone</td></tr><tr><td>Prostaglandin*</td><td>Sigma-Aldrich</td><td>P5172</td><td>0.28 &amp;micro;M prostaglandin</td></tr><tr><td>Putrescine*</td><td>Sigma-Aldrich</td><td>P5780</td><td>182 &amp;micro;M putrescine</td></tr><tr><td>Scalpel Albion</td><td>EMS</td><td>72000</td><td></td></tr><tr><td>Scissor</td><td>Moria</td><td>15396-00</td><td></td></tr><tr><td>Sodium pyruvate*</td><td>Sigma-Aldrich</td><td>S8636</td><td>1 mM sodium pyruvate</td></tr><tr><td>Sodium selenite* (S)</td><td>Sigma-Aldrich</td><td>I1884 (ITS)</td><td>0.29 &amp;micro;M Na&lt;sub&gt;2&lt;/sub&gt;SeO&lt;sub&gt;3&lt;/sub&gt;&amp;nbsp;&amp;nbsp; (S)</td></tr><tr><td>Taurine*</td><td>Sigma-Aldrich</td><td>T8691</td><td>3 mM taurine</td></tr><tr><td>Transferrin* (T)</td><td>Sigma-Aldrich</td><td>I1884 (ITS)</td><td>0.07 &amp;micro;M transferrin (T)</td></tr><tr><td>Vibratome apparatus</td><td>Leica</td><td>VT1000-S</td><td></td></tr><tr><td colspan="4">* Supplements</td></tr></tbody></table>

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.

Apart from transplantation, the photoreceptor layers have also been used to study cell signaling by seeding cells for making photoreceptor cultures12,22. Additionally, they are used to study gene expression and circadian rhythm23,24. We have used the cell photoreceptor layer from a wild-type mouse at post-natal day 8 to prepare photoreceptor cultures in the absence of FCS and maintained the cells for 5 days at 37 &#176;C in an incubator with 5% CO2 (Figure 1E). The cells were then fixed using 4% paraformadehyde and then proceeded for immunocytochemical analysis using methods provided by the antibody suppliers either with mouse monoclonal anti-RHO (1:250, Millipore Mab5316) or rabbit polyclonal anti-SAG (1:200, a generous gift of Igal Gery and David Hicks). We used anti-RHO and anti-SAG (although anti-SAG label cones and rods) due to the fact that anti-SAG is an earlier marker, than anti-RHO. The proportion of cells positive for the anti-RHO antibody is lower than those positive for the anti-SAG antibodies (Figure 2). This can be explained by the fact that SAG, the rod arrestin is also expressed by cones which make 3% of photoreceptors in the isolated layer, or alternatively by the fact that during post-natal maturation of the retina, the expression of SAG precedes that of RHO25,26.
The photoreceptor layer was also used to monitor the specific expression of genes by photoreceptors of the wild-type retina and brain of animals aged 35 days RNA were prepared using CsCl ultracentrifugation 27 and hybridized to mouse DNA chip Array. The messengers for rhodopsin (Rho), S-arrestin (Sag) and cone-transducin (Gnat2) are expressed specifically in the retina as compared to the brain (Figure 3A). The expression is prominent in the photoreceptor layer (PR) than in the whole retina which encompasses the inner retinal layer showing that these genes are expressed indeed by photoreceptors. For Gnat2, the relative expression as compared to Rho and Sag shows that it is expressed by cones contained within the isolated photoreceptor layer. The expression of recoverin (Rcvrn) in the photoreceptor layer as compared to the whole retina is increased (Figure 3B).
The photoreceptor layer was also used to monitor the expression of RHO and GNAT2 using western blotting following methods provided by the antibody suppliers and to compare to the inner retinal layer (Figure 4). Notice the absence of RHO and GNAT228, the markers of rods and cones respectively in the whole retina and in the retina of rd1 mouse at post-natal day 35.
<img alt="Figure 1" src="/files/ftp_upload/51954/51954fig1highres.jpg" /> 
Figure 1. Schematic view of the vibratome sectioning of mouse retina. (A) Installation of the flat-mounted retina with the photoreceptor facing down on the gelatin slice. (B) Section of the inner retina with the vibratome blade. (C) Section of the outer retina added of gelatin. (D) Control of the presence of photoreceptor at the edge of the fragment of retina after the isolation of photoreceptor layer. Scale bar used for green fluorescence is used of a white light microscope. The photoreceptors are located in the edge of the picture. (E) Cultured photoreceptor cells (post-five days). (F) Higher magnification of panel E.
<img alt="Figure 2" src="/files/ftp_upload/51954/51954fig2highres.jpg" width="550" /> 
Figure 2. Differential expression of RHO and SAG in mouse photoreceptor culture. (A) Staining of nuclei (blue), (B) SAG staining (green), (C) RHO staining (red). (D) Merged image. Because staining of SAG is expressed earlier than RHO staining, only two cells are double labeled SAG and RHO (see *). Scale bar: 23 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51954/51954fig2highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/51954/51954fig3highres.jpg" /> 
Figure 3. Comparison of the expression of three genes in brain and whole retina and photoreceptors of the wild-type animals aged thirty five days. (A) Rhodopsin (Rho), S-arrestin (Sag) and cone-transducin (Gnat2). (B) Calbindin (Calb1) and Recoverin (Rcvrn). The data are displayed as relative expression. The relative expression is an expression value obtained from microarray data after normaliszation with the software Robust Multi-array Average (RMA) available in the knowledge database KBaSS (http://kbass.institut-vision.org/KBaSS/).
<img alt="Figure 4" src="/files/ftp_upload/51954/51954fig4highres.jpg" width="400" /> 
Figure 4. Expression of RHO and GNAT2 in outer retina from a wild-type retina at post-natal day 35. Absence of their expression in the inner retina and in the retina of the rd1 mouse at post-natal day 35. ACTB, beta-actin. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51954/51954fig4highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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Vibratome Sectioning Mouse Retina to Prepare Photoreceptor Cultures

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 ...

vibratome-sectioning-mouse-retina-to-prepare-photoreceptor-cultures

Nanyang Technological University

Research

JoVE Journal

Neuroscience

17.7K Views.  UMR_S 968, Institut de la Vision. Neural retina of a mouse aged 8 days is on top of a 4% gelatin block. After isolation of the photoreceptor layer (200 µm) 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.

Video: Vibratome Sectioning Mouse Retina to Prepare Photoreceptor Cultures

Organotypic Culture of Full-thickness Adult Porcine Retina

There is a recognized demand for in vitro models that can replace or reduce animal experiments. Porcine retina has a similar neuronal structure to human retina and is therefore a valuable species for studying mechanisms of human retinal injury and degenerative disease. Here we describe a cost-effective technique for organotypic culture of adult porcine retina isolated from eyes obtained from an abattoir. After removing the anterior segment, a trephine blade was used to create multiple neural retina-Bruch's membrane-RPE-choroid-sclera explants from the posterior segment of adult porcine eyes. A piece of sterile filter paper was used to lift the neural retina off from each explant. The filter paper-retina complex was cultured (photoreceptor side up) atop an insert, which was held away from the bottom of the culture dish by a custom-made stand. The stand allows for good circulation of the culture medium to both sides of the retina. Overall, this procedure is simple, reproducible, and permits preservation of native retinal structure for at least seven days, making it a useful model for a variety of morphological, pharmacological, and biochemical studies on mammalian retina.

Here we describe a cost-effective technique for organotypic culture of adult porcine retina for seven days. Briefly, a sterile filter paper was used to lift the neural retina off from the RPE and place photoreceptor side up on an insert raised by a custom-made stand.

There is a recognized demand for in vitro models that can replace or reduce animal experiments. Porcine retina has a similar neuronal structure to human ...

Quantifying the Activity of cis-Regulatory Elements in the Mouse Retina by Explant Electroporation

Transcription factors within cellular gene networks control the spatiotemporal pattern and levels of expression of their target genes by binding to cis-regulatory elements (CREs), short (&tilde;300-600 bp) stretches of genomic DNA which can lie upstream, downstream, or within the introns of the genes they control. CREs (i.e., enhancers/promoters) typically consist of multiple clustered binding sites for both transcriptional activators and repressors1-3. They serve as logical integrators of transcriptional input giving a unitary output in the form of spatiotemporally precise and quantitatively exact promoter activity. Most studies of mammalian cis-regulation to date have relied on mouse transgenesis as a means of assaying the enhancer function of CREs4-5. This technique is time-consuming, costly and, on account of insertion site effects, largely non-quantitative. On the other hand, quantitative assays for mammalian CRE function have been developed in tissue culture systems (e.g., dual luciferase assays), but the in vivo relevance of these results is often uncertain. 
Electroporation offers an excellent alternative to traditional mouse transgenesis in that it permits both spatiotemporal and quantitative assessment of cis-regulatory activity in living mammalian tissue. This technique has been particularly useful in the analysis of cis-regulation in the central nervous system, especially in the cerebral cortex and the retina6-8. While mouse retinal electroporation, both in vivo and ex vivo, has been developed and extensively described by Matsuda and Cepko6-7,9, we have recently developed a simple approach to quantify the activity of photoreceptor-specific CREs in electroporated mouse retinas10. Given that the amount of DNA that is introduced into the retina by electroporation can vary from experiment to experiment, it is necessary to include a co-electroporated 'loading control' in all experiments. In this respect, the technique is very similar to the dual luciferase assay used to quantify promoter activity in cultured cells. 
When assaying photoreceptor cis-regulatory activity, electroporation is usually performed in newborn mice (postnatal day 0, P0) which is the time of peak rod production11-12. Once retinal cell types become post-mitotic, electroporation is much less efficient. Given the high rate of rod birth in newborn mice and the fact that rods constitute more than 70% of the cells in the adult mouse retina, the majority of cells that are electroporated at P0 are rods. For this reason, rod photoreceptors are the easiest retinal cell type to study via electroporation. The technique we describe here is primarily useful for quantifying the activity of photoreceptor CREs.

Quantifying the Activity of cis-Regulatory Elements in the Mouse Retina by Explant Electroporation

This protocol describes a simple and inexpensive way to quantify the activity of cis-regulatory elements (i.e., enhancer/promoters) in living mouse retinas via explant electroporation. DNA preparation, retinal dissection, electroporation, retinal explant culture, and post-fixation analysis and quantification are described.

Transcription factors within cellular gene networks control the spatiotemporal pattern and levels of expression of their target genes by binding to ...

Recording Light-evoked Postsynaptic Responses in Neurons in Dark-adapted, Mouse Retinal Slice Preparations Using Patch Clamp Techniques

The retina is the gateway to the visual system. To understand visual signal processing mechanisms, we investigate retinal neural network functions. Retinal neurons in the network comprise of numerous subtypes. More than 10 subtypes of bipolar cells, ganglion cells, and amacrine cells have been identified by morphological studies. Multiple subtypes of retinal neurons are thought to encode distinct features of visual signaling, such as motion and color, and form multiple neural pathways. However, the functional roles of each neuron in visual signal processing are not fully understood. The patch clamp method is useful to address this fundamental question. Here, a protocol to record light-evoked synaptic responses in mouse retinal neurons using patch clamp recordings in dark-adapted conditions is provided. The mouse eyes are dark-adapted O/N, and retinal slice preparations are dissected in a dark room using infrared illumination and viewers. Infrared light does not activate mouse photoreceptors and thus preserves their light responsiveness. Patch clamp is used to record light-evoked responses in retinal neurons. A fluorescent dye is injected during recordings to characterize neuronal morphological subtypes. This procedure enables us to determine the physiological functions of each neuron in the mouse retina.

We will demonstrate how to prepare retinal slices from the mouse eye and record light responses in retinal neurons. The entire procedure is conducted in dark-adapted conditions.

The retina is the gateway to the visual system. To understand visual signal processing mechanisms, we investigate retinal neural network functions. ...

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 ...

Preparation of Horizontal Slices of Adult Mouse Retina for Electrophysiological Studies

Vertical slice preparations are well established to study circuitry and signal transmission in the adult mammalian retina. The plane of sectioning in these preparations is perpendicular to the retinal surface, making it ideal for the study of radially oriented neurons like photoreceptors and bipolar cells. However, the large dendritic arbors of horizontal cells, wide-field amacrine cells, and ganglion cells are mostly truncated, leaving markedly reduced synaptic activity in these cells. Whereas ganglion cells and displaced amacrine cells can be studied in a whole-mounted preparation of the retina, horizontal cells and amacrine cells located in the inner nuclear layer are only poorly accessible for electrodes in whole retina tissue.
To achieve maximum accessibility and synaptic integrity, we developed a horizontal slice preparation of the mouse retina, and studied signal transmission at the synapse between photoreceptors and horizontal cells. Horizontal sectioning allows&#160;(1) easy and unambiguous visual identification of horizontal cell bodies for electrode targeting, and (2) preservation of the extended horizontal cell dendritic fields, as a prerequisite for intact and functional cone synaptic input to horizontal cell dendrites.
Horizontal cells from horizontal slices exhibited tonic synaptic activity in the dark, and they responded to brief flashes of light with a reduction of inward current and diminished synaptic activity. Immunocytochemical evidence indicates that almost all cones within the dendritic field of a horizontal cell establish synapses with its peripheral dendrites. The horizontal slice preparation is therefore well suited to study the physiological properties of horizontally extended retinal neurons as well as sensory signal transmission and integration across selected synapses.

We developed a horizontal slice preparation of the adult mammalian retina with the plane of sectioning parallel to the retinal surface. Dendritic fields of nonradially oriented retinal neurons were not truncated, allowing the study of signal processing with patch-clamp and imaging techniques.

Vertical slice preparations are well established to study circuitry and signal transmission in the adult mammalian retina. The plane of sectioning in ...

Optimized Protocol for Retinal Wholemount Preparation for Imaging and Immunohistochemistry

Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ring-supported hydrophilized PTFE membrane to provide structural support to both living and fixed tissue during immunohistochemical processing, which allows for the use of a variety of protocols that would otherwise cause damage to the tissue. First, this is demonstrated with bolus loading of fluorescent markers into living retinal tissue. This method allows for quick visualization of targeted structures, while the membrane support maintains tissue integrity during the injection and allows for easy transfer of the preparation for further imaging or processing.
Second, a procedure for antibody staining in tissue fixed with carbodiimide is described. Though paraformaldehyde fixation is more common, carbodiimide fixation provides a superior substrate for the visualization of synaptic proteins. A limitation of carbodiimide is that the resulting fixed tissue is relatively fragile; however, this is overcome with the use of the supporting membrane. Retinal tissue is used to demonstrate these techniques, but they may be applied to any fragile tissue.

The protocol described here is for structural assessment of a wholemount retinal preparation. This includes descriptions of tissue dissection, mounting onto a hydrophilized polytetrafluoroethylene (PTFE) membrane insert, bolus loading with fluorescent markers, and a comparison of fixation with carbodiimide and paraformaldehyde for immunohistochemical analysis of cellular and synaptic components.

Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ...

Biology

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.

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 ...

Studying Bipolar and Horizontal Neuronal Cells in the Murine Split Retina

Split Retina as an Improved Flatmount Preparation for Studying Inner Nuclear Layer Neurons in Vertebrate Retina

Bipolar cells and horizontal cells of the vertebrate retina are the first neurons to process visual information after photons are detected by photoreceptors. They perform fundamental operations such as light adaptation, contrast sensitivity, and spatial and color opponency. A complete understanding of the precise circuitry and biochemical mechanisms that govern their behavior will advance visual neuroscience research and ophthalmological medicine. However, current preparations for examining bipolar and horizontal cells (retinal whole mounts and vertical slices) are limited in their capacity to capture the anatomy and physiology of these cells. In this work, we present a method for removing photoreceptor cell bodies from live, flatmount mouse retinas, providing enhanced access to bipolar and horizontal cells for efficient patch clamping and rapid immunolabeling. Split retinas are prepared by sandwiching an isolated mouse retina between two pieces of nitrocellulose, then gently peeling them apart. The separation splits the retina just above the outer plexiform layer to yield two pieces of nitrocellulose, one containing the photoreceptor cell bodies and another containing the remaining inner retina. Unlike vertical retina slices, the split retina preparation does not sever the dendritic processes of inner retinal neurons, allowing for recordings from bipolar and horizontal cells that integrate the contributions of gap junction-coupled networks and wide-field amacrine cells. This work demonstrates the versatility of this preparation for the study of horizontal and bipolar cells in electrophysiology, immunohistochemistry, and in situ hybridization experiments.

Author Spotlight: Using the Split Retina Technique for Enhanced Access and Accelerated Experiments 

This work presents an alternative flatmount retina preparation in which the removal of photoreceptor cell bodies enables faster antibody diffusion and improved patch pipette access to inner retinal neurons for immunohistochemistry, in situ hybridization, and electrophysiology experiments.

Bipolar cells and horizontal cells of the vertebrate retina are the first neurons to process visual information after photons are detected by ...

Characterization and Morphological Dynamics of Cultured Primary Murine RPE

A Simplified Method for Isolation and Culture of Retinal Pigment Epithelial Cells from Adult Mice

Retinal pigment epithelial cells (RPE) are critical for the proper function of the retina. RPE dysfunction is involved in the pathogenesis of important retinal diseases, such as age-related macular degeneration, retinitis pigmentosa, and diabetic retinopathy. We present a streamlined approach for the isolation of RPE from murine adult eyes. In contrast to previously reported methods, this approach enables the isolation and culture of highly pure RPE from adult mice. This simple and fast method does not require extensive technical skill and is achievable with basic scientific tools and reagents. Primary RPE are isolated from C57BL/6 background mice aged 3- to 14-weeks by enucleation of the eye followed by the removal of the anterior segment. Enzymatic trypsinization and centrifugation are used to dissociate and isolate the RPE from the eyecup. In conclusion, this approach offers a quick and effective protocol for the utilization of RPE in the study of retinal function and disease.

Author Spotlight: Efficient Isolation of Primary Retinal Pigment Epithelial Cells from Adult Mice for Retinopathy Research

Retinal pigment epithelium (RPE) acts as a crucial barrier between the choroid and retina, promoting the health and function of retinal cell types, such as photoreceptors. Herein, we describe a simple and effective protocol for isolating and culturing adult murine RPE.

Retinal pigment epithelial cells (RPE) are critical for the proper function of the retina. RPE dysfunction is involved in the pathogenesis of important ...

Cryosectioning and Immunostaining of Mouse Retina

Tissue sectioning and immunohistochemistry are essential techniques in histological and pathological studies of retinal diseases using animal models. These methods enable detailed examinations of tissue morphologies and the localization of specific proteins within the tissue, which provide valuable insights into disease processes and mechanisms. Mice are the most widely used model for this purpose. However, because mouse eyeballs are small and mouse retinas are extremely delicate tissues, obtaining high-quality retinal sections and immunostaining images from mouse eyeballs is typically challenging. This study describes an improved protocol for cryosectioning mouse retinas and performing immunohistochemistry. An essential point of this protocol involves coating the eyeball with a layer of super glue, which prevents deformation of the eyeballs during the processes of cornea removal, lens extraction, and embedding. This step ensures the integrity of retinal morphologies is well preserved. This protocol highlights critical technical considerations and optimization strategies for consistently producing high-quality retinal sections and achieving excellent immunostaining results.

A protocol for preparing mouse retinal cryosections and performing immunostaining on photoreceptors is described. This article enables researchers to consistently produce mouse retinal frozen sections with well-preserved morphology and high-quality immunostaining results.

Tissue sectioning and immunohistochemistry are essential techniques in histological and pathological studies of retinal diseases using animal models. ...

Vibratome Sectioning Mouse Retina to Prepare Photoreceptor Cultures

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Przeglądaj więcej filmów

Organotypic Culture of Full-thickness Adult Porcine Retina

Quantifying the Activity of cis-Regulatory Elements in the Mouse Retina by Explant Electroporation

Recording Light-evoked Postsynaptic Responses in Neurons in Dark-adapted, Mouse Retinal Slice Preparations Using Patch Clamp Techniques

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

Preparation of Horizontal Slices of Adult Mouse Retina for Electrophysiological Studies

Optimized Protocol for Retinal Wholemount Preparation for Imaging and Immunohistochemistry

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

Split Retina as an Improved Flatmount Preparation for Studying Inner Nuclear Layer Neurons in Vertebrate Retina

A Simplified Method for Isolation and Culture of Retinal Pigment Epithelial Cells from Adult Mice

Cryosectioning and Immunostaining of Mouse Retina