This work was supported by the National Science Foundation Graduate Research Fellowship Program (NSF-GRFP) grant DGE - 1644869 and the National Institute of Neurological Disorders and Stroke (NINDS) of the National Institutes of Health (NIH), award number F99NS124180 NIH NINDS Diversity Specialized F99 (to CKCO), the National Eye Institute (NEI) 5T32EY013933 (to AMP), the National Institute of Neurological Disorders and Stroke (RO1 NS081333, R03 NS099920 to CMT), and the Department of Defense Army/Air Force (DURIP to CMT).

This protocol follows the Association for Research in Vision and Ophthalmology (ARVO) statement for the use of animals in ophthalmic and vision research. Rodent experiments were approved and monitored by the Institutional Animal Care and Use Committee (IACUC) of Columbia University.
1. Preparation of retinal tissue and cryosectioning
<ol>
	<li>Euthanize the animals by administration of intraperitoneal anesthesia (ketamine (80-100 mg/kg) and xylazine (5-10 mg/kg)), and perfuse the mice subjected to RVO (see8 for details) with 4% paraformaldehyde (PFA) using a peristaltic pump. 
	NOTE: Toe-pinch the animal to confirm depth of anesthesia before proceeding with euthanization and perfusion. Details about perfusion can be found in16.</li>
	<li>Harvest the eyes by careful enucleation using forceps and put the globe in 1 mL of 4% PFA. Leave the eyes overnight at 4 &#176;C. Wash the eyes three times for 10 min, adding 1 mL of 1x PBS and placing them in the shaker.</li>
	<li>Immerse the eyes in 1 mL of 30% sucrose for 3 days, until they settle to the bottom of tube, indicating absorption of the sucrose.</li>
	<li>Fill the eyes with optimal cutting temperature compound using a 30 G syringe, until the eye has a rounded appearance (approximately 50 &#181;L).</li>
	<li>Embed the eyes in a cryomold with optimal cutting temperature compound until the eyes are covered and freeze at -80 &#176;C until ready to cryosection.</li>
	<li>Section embedded the eyes at 20 &#181;m onto glass slides using a cryostat.
	<ol>
		<li>Remove the optimal cutting temperature compound block from the cryomold.</li>
		<li>Place it on the cryostat chuck by adding optimal cutting temperature compound and placing the block on the chuck. Leave it inside the cryostat until it freezes.</li>
		<li>Proceed to cut the block at 20 &#181;m until the retinal tissue is seen. 
		NOTE: This can be confirmed with a light microscope at 30x magnification (3x objective, 10x eyepieces). Tissues can be distinguished from the OCT media by color and shape.</li>
		<li>Collect the retinal tissue in microscope slides in a series of four sections per slide. 
		NOTE: See Figure 1A for suggested placing of retinal sections.</li>
	</ol>
	</li>
	<li>Label the slides with an ID that de-identifies the treatment and genotype.</li>
	<li>Store the slides at -20 &#176;C until staining.</li>
</ol>
2. Immunohistochemistry
NOTE: Use fixed cryopreserved tissue for immunohistochemistry to maintain cell morphology. Pick sections that are at the level of the optical coherence tomography (OCT) images acquired in vivo7. Use the first two series of slides collected from the cryostat or sections from 150 &#181;m into the retinal tissue.
<ol>
	<li>Place the slides in a dark and humid slide chamber.</li>
	<li>Permeabilization and blocking: Wash the retinal cross-sections with 300 &#181;L of 1x PBS for 5 min to remove the OCT and discard after.</li>
	<li>Permeabilize the tissue by adding 300 &#181;L of 1x PBS with 0.1% Triton X-100 for 2 h at room temperature (RT) and discard after.</li>
	<li>Block the tissue by adding 300 &#181;L of blocking buffer (10% Normal Goat Serum (NGS), 1% Bovine Serum Albumin (BSA) in 1x PBS, filtered) and leave overnight at 4 &#176;C.</li>
	<li>Primary antibodies: Dilute primary antibodies in blocking buffer. Primary antibodies used include anti-cl-caspase-9 at 1:800, anti-CD31 at 1:50, and anti-caspase-7-488 (direct labeled antibody) at 1:150. 
	NOTE: Follow manufacturer&#39;s recommendation for the appropriate dilution of primary antibodies.
	<ol>
		<li>Pour the blocking buffer off sections and apply 100 &#181;L of the primary antibody cocktail to the retinal slides.</li>
		<li>Incubate the cross-sections overnight at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Wash the sections four times for 5 min with 300 &#181;L of 1x PBS. 
	NOTE: If the tissue sections are very fragile, washes should be removed using capillary action by applying a tissue to the corner of the slide to avoid displacing the retinal sections.</li>
	<li>To avoid cross-labeling of primary antibodies raised in the same host species, complete the staining with secondary antibodies prior to applying the directly labeled antibodies.</li>
	<li>Secondary antibodies: Dilute the secondary antibodies in blocking buffer at a concentration of 0.1%. For example, to detect anti-cl-caspase-9, an antibody raised in rabbit, use goat-anti-rabbit-568 secondary.
	<ol>
		<li>Apply 200 &#181;L of the secondary antibody cocktail to the retinas.</li>
		<li>Incubate the tissue for 2 h at RT.</li>
		<li>Wash the sections four times for 5 min with 300 &#181;L of 1x PBS.</li>
		<li>Stain the nuclei for 5 min with 300 &#181;L of DAPI at a dilution of 0.02%.</li>
	</ol>
	</li>
	<li>Wash once for 5 min with 300 &#181;L of 1x PBS.</li>
	<li>Place a coverslip on the retinal sections using 500 &#181;L of fluoromount-G media, which preserves the fluorescent signal, and carefully place the coverslip on the top of the slide, avoiding bubbles.</li>
</ol>
3. Confocal imaging
<ol>
	<li>Acquire images of the stained sections with confocal microscopy.
	<ol>
		<li>Turn on the confocal microscope.</li>
		<li>Place the slide on the stage.</li>
		<li>Adjust the focus to see the retinal section clearly. 
		NOTE: Take at least four images per section and image four sections per retina. See the Retinal imaging schematic for suggested imaging areas (Figure 1B, C).</li>
	</ol>
	</li>
	<li>Image caspase, vascular, and nuclear staining using a confocal microscope that has Z-stack acquisition, using a 20x or 40x objective. 
	NOTE: The imaging parameters and software setup should be constant for all imaging in an experiment. The 20x objective provides an overview of the retinal layers, which are well-defined by the nuclear staining. The 40x objective provides more cellular detail.
	<ol>
		<li>Set appropriate exposure and laser intensity times per channel by clicking acquire settings.</li>
		<li>Set the Z-stack to visualize the whole section by clicking Z-series and set up and down parameters to cover the depth of the tissue.</li>
	</ol>
	</li>
	<li>Save the images following the masked ID assigned during cryosectioning.</li>
</ol>
4. Quantification of caspase levels
<ol>
	<li>Drag image files of 405, 470, 555, and 640 channels to FIJI&#39;s console.</li>
	<li>Click Image &#62; Color &#62; Merge Channels.</li>
	<li>Assign colors to the files as follows: red to 555, green to 470, gray to 405, cyan to 640.</li>
	<li>Compress the Z-stack by clicking Image &#62; Stack &#62; Z Project.</li>
	<li>Click Projection Type: Max Intensity.</li>
	<li>Open the Channels Interface by clicking Image &#62; Color Channels Tool.</li>
	<li>Open the Brightness and Contrast interface.</li>
	<li>Select parameters per channel.
	<ol>
		<li>Go to Channels and select one channel.</li>
		<li>Put the cursor on top of the caspase expressed cell and annotate the pixel value.</li>
		<li>Put the cursor on top of the background and annotate the pixel value.</li>
	</ol>
	</li>
	<li>Test parameters
	<ol>
		<li>Go to the Brightness and Contrast&#160;interface.</li>
		<li>Click Select.</li>
		<li>Plug in the minimum displayed value (annotated pixel value of the caspase expressed cell).</li>
		<li>Plug in the maximum displayed value (annotated pixel value of the background).</li>
		<li>Click Ok.</li>
	</ol>
	</li>
	<li>Repeat steps 4.8-4.9 for all channels.</li>
	<li>Test the parameters by choosing random images from blinded tissue. 
	NOTE: Edit brightness and contrast parameters only if the background value is not adequate for other images.</li>
	<li>Once the parameters are set, open the image files and compress the Z-stack.</li>
	<li>Add in the brightness and contrast parameters for the caspase channel and the isolectin, vascular marker channel.</li>
	<li>Use the point tool to quantify the number of vascular positive areas using colocalization of the vascular marker with caspase expression as the readout of positive areas.</li>
	<li>Repeat step 4.14 but using Hoechst as the marker of neuronal positive areas.</li>
	<li>Annotate the values on a spreadsheet per image.</li>
	<li>Average the values by section.</li>
	<li>Average the section values-this will be the readout per eye.</li>
</ol>
5. Genetic confirmation of the relevance of the endothelial cell caspase-9
<ol>
	<li>Use retinas obtained from the Casp9FL/FL-VECad-CreERT2 mice that were subjected to RVO7.</li>
	<li>Immunostain the retinas for caspase-9 (the deleted caspase), caspase-7 (a down-stream effector caspase), vascular markers, and DAPI as described above in section 2.</li>
	<li>Image and quantify as described above in sections 3 and 4.</li>
</ol>
6. Targeting caspase-9 in RVO
<ol>
	<li>Obtain eyes from the mice subjected to RVO followed by application of the caspase-9 inhibitor Pen1-XBir3 topically to eyes as in7.</li>
	<li>Immunostain the retinas for caspase-7 (a down-stream effector caspase), vascular markers, and DAPI as described above in section 2.</li>
	<li>Image and quantify as described above in sections 3 and 4.</li>
</ol>

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The authors declare the following competing interests: C.M.T. has the following patent applications US20200164026, US20190142915, and US20150165061. C.M.T. and S.S. have a patent application US 20140024597. C.M.T., A.M.P., and M.I.A. have a patent application US2020058683. C.M.T. and Y.Y.J. have a patent application WO2018013519. M.I.A and C.M.T are listed as inventors on a patent application WO/2020/223212 by the Trustees of Columbia University in the City of New York. The remaining authors declare no competing interests.

Caspases are a multi-membered family of proteases best studied for their roles in cell death and inflammation; however, more recently a variety of non-death functions have been uncovered for some family members4,5. Much of our understanding of caspase function is derived from work in cell culture and from inferential data from human disease. While it is appreciated that there is aberrant induction, activation, or inactivation of caspases in disease, it has been challenging to functionally determine if the caspases are driving disease pathology. Many tools used to assess specific caspases detect multiple family members, undercutting the functional relevance of data obtained with these reagents17. Here we provide a protocol to study caspases in complex tissue, using the retina as an example. In the nervous system, caspase expression is down-regulated pre- and postnatally. This allows the use of specific antibodies to test changes in caspase levels in a disease model.
While the focus of the protocol is on image processing and analysis, the success of the technique also relies on careful tissue preparation, as well as IHC and microscopic imaging to ensure consistency, validity, and reliability of the data. Care must be taken to utilize the same parameters to image an entire data set. Poor quality images, low contrast, and blurry focus will not provide reliable data. When sectioning and imaging P-RVO regions of the retina, tears and folding should be avoided as these areas may result in artifacts. Areas of direct damage should also be avoided. The investigator performing the imaging needs to be blinded to the treatment of the tissue. For analysis, it is recommended that two independent observers, blinded to the treatment group, perform the analysis. In addition, primary mouse antibodies should not be used in mouse injury models as the secondary antibodies bind non-specifically to the vasculature. An alternative is to use directly conjugated primary antibodies.
An alternative approach to cell counting is quantification of the percentage of the area of expression. This can be used when cell counting is not possible (e.g., if caspase expression is localized to neuronal processes or photoreceptor segments). Central and peripheral retina are differentially affected in various injury models. The method can be adapted to specifically analyze different retinal regions. If performing BRVO, then sections should be selected from the injured portion of the eye. The protocol also provides for staining of different cell markers to identify where the caspase is expressed.
Limitations of the method include that it does not differentiate between high and low levels of caspase signal in a given cell. Additionally, using a nuclear stain to identify neuronal layers is convenient but does not definitively indicate neuronal expression (glia and leukocytes are present in these layers also). Additional neuronal markers can be used to validate neuronal expression. There are also a variety of antibodies available to evaluate caspase expression; these have been best defined for human caspase-9, where there are antibodies for full length/cleaved (cl) caspase-9, autocleaved caspase-9, and caspase-3-cleaved caspase-9.
The mean intensity of the IHC signal is frequently reported as a quantification method. However, background noise or autofluoresence from red blood cells can produce inaccurate results. Cell specific quantification allows for determination and discrimination of background, non-specific staining, and autofluoresence.
This protocol can be used to measure tissue wide changes in the expression of individual caspases, changes in expression in a specific cell type, such as an endothelial cell, and changes in specific cell types in specific locations, such as neurons in different retinal layers. This flexibility allows the experimenter to query how the disease state is altering individual caspase levels. Existing/alternative methods use western blotting analysis for semiquantitative comparison of caspase signaling, although methods will not provide the clear separation of cellular localization that this protocol acheives. Notably, in Figure 3D, caspase-9 inhibition is more effective at reducing neuronal caspase-9 in the INL and ONL, compared to the RGL. This kind of resolution is challenging to achieve by other methods. Additionally, when a whole tissue is analyzed biochemically, the levels of caspases that change may be too low to pick up, since there are many different cell types in a complex tissue.
The antibodies, which must be validated by the user, provide specificity for the caspase probed. Caspases can act in cascades (i.e., initiator activating effector and then leading to outcome - death, inflammation, cell signaling). This protocol can be used in samples collected at different time points post-damage; in the examples presented here, samples are collected at different times post-RVO. In previously published work, it has been found that caspase-9 was increased within 1 h post-RVO7, which lead to further validation and targeting studies. After identifying endothelial caspase-9 as a potential driver of RVO pathology, a mouse with inducible endothelial cell caspase-9KO to validate endothelial caspase-9 was used. The use of inducible cell specific caspase KO mice provides another level of specificity and avoids the developmental death seen with constitutive caspase-9KO18. It also avoids compensatory changes in other family members that have been shown to occur in constitutive caspase KO mice19. Moreover, the use of cell specific inducible caspase KO mice allows the identification of the cell type in which the caspase is regulating pathology in the tissue. The protocol also provides the steps to interrogate the efficacy of a therapeutic approach, targeting active caspase-9 in RVO. This approach can also be applied to other tissues, including the brain.

The caspases are a family of proteases that regulate developmental cell death, immune responses, and aberrant cell death in disease1,2. While it is well known that members of the caspase family are induced in a variety of neurodegenerative diseases, understanding which caspase drives disease pathology is more challenging3. Such studies require tools to identify, characterize, and validate the function of individual caspase family members. Parsing out the relevant individual caspases is important both from a mechanistic and a therapeutic standpoint, as the literature has multiple studies providing evidence of the diverse roles of caspases4,5. Thus, if the goal is to target a caspase in a disease for a therapeutic benefit, it is critical to have specific targeting of the relevant family member(s). Traditional techniques to detect caspase levels in tissue include western blotting and enzymatic and fluorometric approaches3,6. However, none of these measures allow for cell-specific detection of caspase levels, and in some scenarios, cleaved caspases often cannot be detected by traditional protein analysis measures. It is known that caspases can play different apoptotic and non-apoptotic roles in the same tissue7, therefore careful characterization of cell-specific caspase levels is needed for accurate understanding of developmental and disease pathways.
This study shows caspase activation and function in a model of neurovascular hypoxia-ischemia - retinal vein occlusion (RVO)7,8. In a complex tissue such as the retina, there are multiple cell types that can be affected by the hypoxia-ischemia induced in RVO, including glial cells, neurons, and vasculature7. In the adult mouse retina, there is very little expression of caspases evident in healthy tissue, as measured by immunohistochemistry (IHC)7, but that is not the case during development9 or in models of retinal disease10,11. IHC is a technique that is well established in biomedical research and has allowed validation of disease and pathological targets, identification of new roles through spatial localization, and quantification of proteins. In cases where cleaved caspase products cannot be detected by western blot or fluorometric analysis, nor the specific cell location of distinct caspases or interrogation of caspase signaling pathways through localization, then IHC should be used.
In order to determine the caspase(s) functionally relevant in RVO, IHC was used with validated antibodies for caspases and cellular markers. The previous studies performed in the lab showed that caspase-9 was rapidly activated in a model of ischemic stroke and inhibition of caspase-9 with a highly specific inhibitor protected from neuronal dysfunction and death12. Because the retina is part of the central nervous system (CNS), it serves as a model system to query and further investigate the role of caspase-9 in neurovascular injuries13. To this end, the mouse model of RVO was used to study the cell-specific location and distribution of caspase-9 and its implication in neurovascular injury. RVO is a common cause of blindness in working aged adults that results from vascular injury14. It was found that caspase-9 was expressed in a non-apoptotic manner in endothelial cells, but not in neurons.
As a tissue, the retina has the advantage of being visualized as either a flatmount, which allows appreciation of the vascular networks, or as cross-sections, which highlights the neuronal retinal layers. Quantification of caspase protein expression in cross-sections provides context, regarding which caspase is potentially critical in retinal neuronal connectivity and vision function by identifying the localization of the caspase(s) in the retina. After identification and validation, targeting of the caspase of interest is achieved using inducible cell specific deletion of the caspase identified. For potential therapeutic inquiries, the relevance of the caspases of interest was tested using specific tools to inhibit the activated caspase. For caspase-9 a cell permeant highly selective inhibitor7,15, Pen1-XBIR3 was used. For this report, 2-month-old, male C57BL/6J strain and tamoxifen-inducible endothelial caspase-9 knockout (iEC Casp9KO) strain with a C57BL/6J background were used. These animals were exposed to the mouse model of RVO and C57BL/6J were treated with the caspase-9 selective inhibitor, Pen1-XBir3. The described methodology can be applied to other models of disease in the central and peripheral systems7,15.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>anti-Caspase-7 488</td>
			<td>Novus Biologicals</td>
			<td>NB-56529AF488</td>
			<td>use at 1:150</td>
		</tr>
		<tr>
			<td>anti-cl-Caspase-9</td>
			<td>Cell Signaling</td>
			<td>9505-S</td>
			<td>use at 1:800</td>
		</tr>
		<tr>
			<td>anti-CD31</td>
			<td>BD Pharmingen</td>
			<td>553370</td>
			<td>use at 1:50</td>
		</tr>
		<tr>
			<td>Confocal Spinning Disc Microscope</td>
			<td>Biovision</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FIJI 2.3.0</td>
			<td>open source</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fluormount G</td>
			<td>Fisher</td>
			<td>50-187-88</td>
			<td></td>
		</tr>
		<tr>
			<td>Forcep</td>
			<td>Roboz</td>
			<td>RS-5015</td>
			<td></td>
		</tr>
		<tr>
			<td>iCasp9FL/FL X VECad-CreERT2 mice</td>
			<td>lab generated</td>
			<td></td>
			<td>see Avrutsky 2020</td>
		</tr>
		<tr>
			<td>Isolectin (594, 649)</td>
			<td>Vector</td>
			<td>DL-1207</td>
			<td>use at 1:200</td>
		</tr>
		<tr>
			<td>Ketamine Hydrochloride</td>
			<td>Henry Schein</td>
			<td>NDC: 11695-0702-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Perfusion pump&#160;</td>
			<td>Masterflex</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pen1-XBir3</td>
			<td>lab generated</td>
			<td></td>
			<td>see Avrutsky 2020</td>
		</tr>
		<tr>
			<td>Prism 9.1</td>
			<td>GraphPad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-Tek O.C.T.</td>
			<td>Fisher</td>
			<td>14-373-65</td>
			<td></td>
		</tr>
		<tr>
			<td>Vis-a-View 4.0</td>
			<td>Visitron Systems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Akorn</td>
			<td>NDCL 59399-110-20</td>
			<td></td>
		</tr>
	</tbody>
</table>

quantification of immunostained caspase-9 in retinal tissue

The family of caspases is known to mediate many cellular pathways beyond cell death, including cell differentiation, axonal pathfinding, and proliferation. Since the identification of the family of cell death proteases, there has been a search for tools to identify and expand the function of specific family members in development, health, and disease states. However, many of the currently commercially available caspase tools that are widely used are not specific for the targeted caspase. In this report, we delineate the approach we have used to identify, validate, and target caspase-9 in the nervous system using a novel inhibitor and genetic approaches with immunohistochemical read-outs. Specifically, we used the retinal neuronal tissue as a model to identify and validate the presence and function of caspases. This approach enables the interrogation of cell-type specific apoptotic and non-apoptotic caspase-9 functions and can be applied to other complex tissues and caspases of interest. Understanding the functions of caspases can help to expand current knowledge in cell biology, and can also be advantageous to identify potential therapeutic targets due to their involvement in disease.

The described protocol allows the user to analyze and quantify caspase-9 levels in the retinal tissue. Additionally, it present tools to further identify, validate, and specifically target caspase-9 and downstream substrates. The summarized steps allow quantifiable analysis of caspase levels and cellular specificity in fluorescent photomicrographs. All figures show representative photomicrographs and quantification of the indicated caspase levels in the total retina, endothelial cells, and neurons in uninjured and 1-day P-RVO retina cross-sections. The retinal cross-section allows visualization of retinal nuclei in the retinal ganglion layer (RGL), inner nuclear layer (INL), and outer nuclear layer (ONL) when stained with Hoechst. In addition, retinal blood vessels are visible in the retinal plexiform layers or between the RGL and INL, and between the INL and ONL. Because of the histological nature of blood vessels, when the retina is sectioned, blood vessels will appear as disconnected and separate, supplying the plexiform layers. Figure 2 identifies caspase-9 as highly regulated 1-day P-RVO. Figure 3 validates the functional relevance of endothelial caspase-9 by using inducible endothelial cell knockout mice. Figure 4 shows that targeting active caspase-9 pharmacologically blocks the induction of a downstream target of caspase-9-caspase-7. The protocol identifies the cellular localization of the caspases and the neuronal retinal layers in which the caspases are expressed.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64237/64237fig01.jpg" /> 
Figure 1: Retinal imaging scheme. (A) Recommended placing of retinal sections in the microscope slide. (B) Overview of the imaging areas in the retina. (C) Representation of retinal view at 20x objective. Retinal layers: RGL = retinal ganglion layer, INL = inner nuclear layer, ONL = outer nuclear layer. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64237/64237fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64237/64237fig02.jpg" /> 
Figure 2: RVO induces caspase-9 in endothelial cells and neurons. (A) Representative retinal cross-sections from uninjured and 1-day P-RVO stained with cl-caspase-9 1:800 (green), isolectin 1:200 (red), and DAPI (white). (B) Quantification of the number of neurons with cl-caspase-9 (see Table of Materials for details on the antibodies). (C) Quantification of the number of endothelial cells with cl-caspase-9. (D) Total number of cells expressing cl-caspase-9. Uninjured, n = 6; 1-day P-RVO, n = 5. Error bars represent the mean &#177; SEM; One-way ANOVA, Fisher&#39;s LSD test. Scale bar = 50 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64237/64237fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64237/64237fig03v3.jpg" /> 
Figure 3: Endothelial cell deletion of endothelial caspase-9 blocks RVO-induction of caspase-7. (A) Representative retinal cross-sections from iEC Casp9WT and iEC Casp9KO littermate mice 1-day P-RVO stained with caspase-7 (green), cl-caspase-9 (blue), CD31, a vascular marker (red), and DAPI (white). (B) Quantification of the number of cells in the inner retina with cl-caspase-9 and with caspase-7. (C) Quantification of the number of endothelial cells with cl-caspase-9 and with caspase-7. (D) Total number of cells expressing cl-caspase-9 or caspase-7. iEC Casp9WT, n = 6-12; iEC Casp9KO, n = 3-8. Error bars represent the mean &#177; SEM; One-way ANOVA, Fisher&#39;s LSD test. Scale bar = 50 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64237/64237fig03largev3.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64237/64237fig04.jpg" /> 
Figure 4: Inhibition of caspase-9 activity inhibits caspase-7 expression P-RVO. (A) Representative retinal cross-sections from uninjured and 1-day P-RVO treated or untreated with Pen1-XBIR3 stained with caspase-7 (green), isolectin (red), and DAPI (white). (B) Quantification of the number of endothelial cells with caspase-7. (C) Quantification of the number of leukocytes with caspase-7. (D) Quantification of the number of neurons per neuronal layer with caspase-7. (E) Total number of cells expressing caspase-7. Uninjured Pen1 Saline, n = 6; uninjured Pen1-XBIR3, n = 5; 1-day P-RVO Pen1 Saline, n = 5; 1-day P-RVO Pen1-XBIR3, n = 3. Error bars represent the mean &#177; SEM; One-way ANOVA, Fisher&#39;s LSD test. Abbreviations: RGL = retinal ganglion layer, INL = inner nuclear layer, and ONL = outer nuclear layer. Scale bar = 50 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64237/64237fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Quantification of Immunostained Caspase-9 in Retinal Tissue at JoVE.com

Quantification of Immunostained Caspase-9 in Retinal Tissue

Presented here is a detailed immunohistochemistry protocol to identify, validate, and target functionally relevant caspases in complex tissues.

The family of caspases is known to mediate many cellular pathways beyond cell death, including cell differentiation, axonal pathfinding, and ...

quantification-of-immunostained-caspase-9-in-retinal-tissue

Nanyang Technological University

Research

JoVE Journal

Neuroscience

1.3K Views.  Columbia University. Presented here is a detailed immunohistochemistry protocol to identify, validate, and target functionally relevant caspases in complex tissues.

Video: Quantification of Immunostained Caspase-9 in Retinal Tissue

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

Morphometric Analyses of Retinal Sections

Morphometric analyses of retinal sections have been used in examining retinal diseases. For examples, neuronal cells were significantly lost in the retinal ganglion cell layer (RGCL) in rat models with N-methyl-D-aspartate (NMDA)&#x2013;induced excitotoxicity1, retinal ischemia-reperfusion injury2 and glaucoma3. Reduction of INL and inner plexiform layer (IPL) thicknesses were reversed with citicoline treatment in rats' eyes subjected to kainic acid-mediated glutamate excitotoxicity4. Alteration of RGC density and soma sizes were observed with different drug treatments in eyes with elevated intraocular pressure3,5,6. Therefore, having objective methods of analyzing the retinal morphometries may be of great significance in evaluating retinal pathologies and the effectiveness of therapeutic strategies.
The retinal structure is multi-layers and several different kinds of neurons exist in the retina. The morphometric parameters of retina such as cell number, cell size and thickness of different layers are more complex than the cell culture system. Early on, these parameters can be detected using other commercial imaging software. The values are normally of relative value, and changing to the precise value may need further accurate calculation. Also, the tracing of the cell size and morphology may not be accurate and sensitive enough for statistic analysis, especially in the chronic glaucoma model. The measurements used in this protocol provided a more precise and easy way. And the absolute length of the line and size of the cell can be reported directly and easy to be copied to other files. For example, we traced the margin of the inner and outer most nuclei in the INL and formed a line then using the software to draw a 90 degree angle to measure the thickness. While without the help of the software, the line maybe oblique and the changing of retinal thickness may not be repeatable among individual observers. In addition, the number and density of RGCs can also be quantified. This protocol successfully decreases the variability in quantitating features of the retina, increases the sensitivity in detecting minimal changes.
 	This video will demonstrate three types of morphometric analyses of the retinal sections. They include measuring the INL thickness, quantifying the number of RGCs and measuring the sizes of RGCs in absolute value. These three analyses are carried out with Stereo Investigator (MBF Bioscience &mdash; MicroBrightField, Inc.). The technique can offer a simple but scientific platform for morphometric analyses.

This video demonstrates three types of morphometric analyses of the retina, which include measuring the inner nuclear layer thickness, quantifying the number of retinal ganglion cells (RGCs) and measuring the sizes of RGCs. The technique can offer a simple but scientific platform for morphometric analyses.

Morphometric analyses of retinal sections have been used in examining retinal diseases. For examples, neuronal cells were significantly lost in the ...

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

The process by which the anterior region of the neural plate gives rise to the vertebrate retina continues to be a major focus of both clinical and basic research. In addition to the obvious medical relevance for understanding and treating retinal disease, the development of the vertebrate retina continues to serve as an important and elegant model system for understanding neuronal cell type determination and differentiation1-16. The neural retina consists of six discrete cell types (ganglion, amacrine, horizontal, photoreceptors, bipolar cells, and M&uuml;ller glial cells) arranged in stereotypical layers, a pattern that is largely conserved among all vertebrates 12,14-18.
While studying the retina in the intact developing embryo is clearly required for understanding how this complex organ develops from a protrusion of the forebrain into a layered structure, there are many questions that benefit from employing approaches using primary cell culture of presumptive retinal cells 7,19-23. For example, analyzing cells from tissues removed and dissociated at different stages allows one to discern the state of specification of individual cells at different developmental stages, that is, the fate of the cells in the absence of interactions with neighboring tissues 8,19-22,24-33. Primary cell culture also allows the investigator to treat the culture with specific reagents and analyze the results on a single cell level 5,8,21,24,27-30,33-39. Xenopus laevis, a classic model system for the study of early neural development 19,27,29,31-32,40-42, serves as a particularly suitable system for retinal primary cell culture 10,38,43-45.
Presumptive retinal tissue is accessible from the earliest stages of development, immediately following neural induction 25,38,43. In addition, given that each cell in the embryo contains a supply of yolk, retinal cells can be cultured in a very simple defined media consisting of a buffered salt solution, thus removing the confounding effects of incubation or other sera-based products 10,24,44-45.
However, the isolation of the retinal tissue from surrounding tissues and the subsequent processing is challenging. Here, we present a method for the dissection and dissociation of retinal cells in Xenopus laevis that will be used to prepare primary cell cultures that will, in turn, be analyzed for calcium activity and gene expression at the resolution of single cells. While the topic presented in this paper is the analysis of spontaneous calcium transients, the technique is broadly applicable to a wide array of research questions and approaches (Figure 1).

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

Xenopus laevis provides an ideal model system for studying cell fate specification and physiological function of individual retinal cells in primary cell culture. Here we present a technique for dissecting retinal tissues and generating primary cell cultures that are imaged for calcium activity and analyzed by in situ hybridization.

The process by which  the anterior region of the neural plate gives rise to the vertebrate retina  continues to be a major focus of both clinical and ...

Quantification of Filamentous Actin (F-actin) Puncta in Rat Cortical Neurons

Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich structures suggest alterations in synaptic integrity and connectivity. Here we provide a detailed protocol for culturing primary rat cortical neurons, Phalloidin staining for F-actin puncta, and subsequent quantification techniques. First, the frontal cortex of E18 rat embryos are dissociated into low-density cell culture, then the neurons grown in vitro for at least 12-14 days. Following experimental treatment, the cortical neurons are stained with AlexaFluor 488 Phalloidin (to label the dendritic F-actin puncta) and microtubule-associated protein 2 (MAP2; to validate the neuronal cells and dendritic integrity). Finally, specialized software is used to analyze and quantify randomly selected neuronal dendrites. F-actin rich structures are identified on second order dendritic branches (length range 25-75 &#181;m) with continuous MAP2 immunofluorescence. The protocol presented here will be a useful method for investigating changes in dendritic synapse structures subsequent to experimental treatments.

Quantification of Filamentous Actin F-actin Puncta in Rat Cortical Neurons

Filamentous actin (F-actin) plays an important role in spinogenesis, synaptic plasticity, and synaptic stability. Quantification of F-actin puncta is therefore a useful tool to study the integrity of synaptic structures. This protocol describes the procedures of quantifying F-actin puncta labeled with Phalloidin in low-density primary cortical neuronal cultures.

Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich ...

Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, survival, and glial communications. To date, numerous studies have reported that defects in these systems cause various neuronal diseases. Thus, understanding the mechanisms underlying vesicle dynamics may provide influential clues that could aid in the treatment of several neuronal disorders. Here, we describe a method for quantifying vesicle motilities, such as motility distance and rate of movement, using a software plug-in for the ImageJ platform. To obtain images for quantification, we labeled neuronal endosome-lysosome structures with EGFP-tagged vesicle marker proteins and observed the movement of vesicles using a time-lapse microscopy. This method is highly useful and simplify measuring vesicle motility in neurites, such as axons and dendrites, as well as in the soma of both neurons and glial cells. Furthermore, this method can be applied to other cell lines, such as fibroblasts and endothelial cells. This approach could provide a valuable advancement of our understanding of membrane trafficking.

The investigation of membrane trafficking is crucial for understanding neuronal functions. Here, we introduce a method for quantifying vesicle motility in neurons. This is a convenient method that can be adapted to the quantification of membrane trafficking in the nervous system.

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, ...

Purification of H3 and H4 Histone Proteins and the Quantification of Acetylated Histone Marks in Cells and Brain Tissue

In all eukaryotic organisms, chromatin, the physiological template of all genetic information, is essential for heredity. Chromatin is subject to an array of diverse posttranslational modifications (PTMs) that mostly occur in the amino termini of histone proteins (i.e., histone tail) and regulate the accessibility and functional state of the underlying DNA. Histone tails extend from the core of the nucleosome and are subject to the addition of acetyl groups by histone acetyltransferases (HATs) and the removal of acetyl groups by histone deacetylases (HDACs) during cellular growth and differentiation. Specific acetylation patterns on lysine (K) residues on histone tails determine a dynamic homeostasis between transcriptionally active or transcriptionally repressed chromatin by (1) influencing the core histone assembly and (2) recruiting synergistic or antagonistic chromatin-associated proteins to the transcription site. The fundamental regulatory mechanism of the complex nature of histone tail PTMs influences the majority of chromatin-templated processes and results in changes in cell maturation and differentiation in both normal and pathological development. The goal of the current report is to provide novices with an efficient method to purify core histone proteins from cells and brain tissue and to reliably quantify acetylation marks on histones H3 and H4.

The purpose of this article is to provide a comprehensive, systematic guide to the efficient purification of histones H3 and H4 and the quantification of acetylated histone residues.

In all eukaryotic organisms, chromatin, the physiological template of all genetic information, is essential for heredity. Chromatin is subject to an array ...

Preparation of Mouse Retinal Cryo-sections for Immunohistochemistry

Preparation of high-quality mouse eye sections for immunohistochemistry (IHC) is critical for assessing the retinal structure and function and for determining the mechanisms underlying retinal diseases. Maintaining structural integrity throughout the tissue preparation is vital for obtaining reproducible retinal IHC data but can be challenging due to the fragility and complexity of retinal cytoarchitecture. Strong fixatives like 10% formalin or Bouin's solution optimally preserve the retinal structure, they often impede IHC analysis by enhancing the background fluorescence and/or diminishing antibody-epitope interactions, a process known as epitope masking. Milder fixatives, on the other hand, like 4% paraformaldehyde, reduces background fluorescence and epitope-masking, meticulous dissection techniques must be utilized to preserve the retinal structure. In this article, we present a comprehensive method to prepare mouse ocular posterior cups for IHC that is sufficient to preserve most antibody-epitope interactions without loss of retinal structural integrity. We include representative IHC with antibodies to various retinal cell type markers to illustrate tissue preservation and orientation under optimal and sub-optimal conditions. Our goal is to optimize IHC studies of the retina by providing a complete protocol from ocular posterior cup dissection to IHC.

This report describes comprehensive methods for preparing frozen mouse retina sections for immunohistochemistry (IHC). Methods described include dissection of the ocular posterior cup, paraformaldehyde fixation, embedding in Optimal Cutting Temperature (OCT) media and tissue orientation, sectioning and immunostaining.

Preparation of high-quality mouse eye sections for immunohistochemistry (IHC) is critical for assessing the retinal structure and function and for ...

Subretinal Transplantation of Human Embryonic Stem Cell-Derived Retinal Tissue in a Feline Large Animal Model

Retinal degenerative (RD) conditions associated with photoreceptor loss such as age-related macular degeneration (AMD), retinitis pigmentosa (RP) and Leber Congenital Amaurosis (LCA) cause progressive and debilitating vision loss. There is an unmet need for therapies that can restore vision once photoreceptors have been lost. Transplantation of human pluripotent stem cell (hPSC)-derived retinal tissue (organoids) into the subretinal space of an eye with advanced RD brings retinal tissue sheets with thousands of healthy mutation-free photoreceptors and has a potential to treat most/all blinding diseases associated with photoreceptor degeneration with one approved protocol. Transplantation of fetal retinal tissue into the subretinal space of animal models and people with advanced RD has been developed successfully but cannot be used as a routine therapy due to ethical concerns and limited tissue supply. Large eye inherited retinal degeneration (IRD) animal models are valuable for developing vision restoration therapies utilizing advanced surgical approaches to transplant retinal cells/tissue into the subretinal space. The similarities in globe size, and photoreceptor distribution (e.g., presence of macula-like region area centralis) and availability of IRD models closely recapitulating human IRD would facilitate rapid translation of a promising therapy to the clinic. Presented here is a surgical technique of transplanting hPSC-derived retinal tissue into the subretinal space of a large animal model allowing assessment of this promising approach in animal models.

Presented here is a surgical technique for transplanting human pluripotent stem cell (hPSC)-derived retinal tissue into the subretinal space of a large animal model.

Retinal degenerative (RD) conditions associated with photoreceptor loss such as age-related macular degeneration (AMD), retinitis pigmentosa (RP) and ...

A Hydrophobic Tissue Clearing Method for Rat Brain Tissue

Hydrophobic tissue clearing methods are easily adjustable, fast, and low-cost procedures that allows for the study of a molecule of interest in unaltered tissue samples. Traditional immunolabeling procedures require cutting the sample into thin sections, which restricts the ability to label and examine intact structures. However, if brain tissue can remain intact during processing, structures and circuits can remain intact for the analysis. Previously established clearing methods take significant time to completely clear the tissue, and the harsh chemicals can often damage sensitive antibodies. The iDISCO method quickly and completely clears tissue, is compatible with many antibodies, and requires no special lab equipment. This technique was initially validated for the use in mice tissue, but the current protocol adapts this method to image hemispheres of control and transgenic rat brains. In addition to this, the present protocol also makes several adjustments to preexisting protocol to provide clearer images with less background staining. Antibodies for Iba-1 and tyrosine hydroxylase were validated in the HIV-1 transgenic rat and in F344/N control rats using the present hydrophobic tissue clearing method. The brain is an interwoven network, where structures work together more often than separately of one another. Analyzing the brain as a whole system as opposed to a combination of individual pieces is the greatest benefit of this whole brain clearing method.

Here we present a hydrophobic tissue clearing method that allows for the viewing of target molecules as part of intact brain structures. This technique has now been validated for F344/N control and HIV-1 transgenic rats of both sexes.

Hydrophobic tissue clearing methods are easily adjustable, fast, and low-cost procedures that allows for the study of a molecule of interest in unaltered ...

Imaging and Quantification of Intact Neuronal Dendrites via CLARITY Tissue Clearing

Brain activity, the electrochemical signals passed between neurons, is determined by the connectivity patterns of neuronal networks, and from the morphology of processes and substructures within these neurons. As such, much of what is known about brain function has arisen alongside developments in imaging technologies that allow further insight into how neurons are organized and connected in the brain. Improvements in tissue clearing have allowed for high-resolution imaging of thick brain slices, facilitating morphological reconstruction and analyses of neuronal substructures, such as dendritic arbors and spines. In tandem, advances in image processing software provide methods of quickly analyzing large imaging datasets. This work presents a relatively rapid method of processing, visualizing, and analyzing thick slices of labeled neural tissue at high-resolution using CLARITY tissue clearing, confocal microscopy, and image analysis. This protocol will facilitate efforts toward understanding the connectivity patterns and neuronal morphologies that characterize healthy brains, and the changes in these characteristics that arise in diseased brain states.

Neuronal dendritic morphology often underlies function. Indeed, many disease processes that affect the development of neurons manifest with a morphological phenotype. This protocol describes a simple and powerful method for analyzing intact dendritic arbors and their associated spines.