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>

<ol><li>Van Opdenbosch, N., Lamkanfi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Caspases+in+cell+death%2C+inflammation%2C+and+disease%5BTitle%5D)+AND+%22Immunity%22%5BJournal%5D)">Caspases in cell death, inflammation, and disease.</a> Immunity. 50 (6), 1352-1364 (2019).</li>
<li>Ramirez, M. L. G., Salvesen, G. S. A primer on caspase mechanisms. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aRamirez%2C+MLG+%22Seminars+in+Cell+%26+Developmental+Biology%22">Seminars in Cell & Developmental Biology</a>. 82, Academic Press. 79-85 (2018).</li>
<li>Troy, C. M., Jean, Y. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Caspases%3A+therapeutic+targets+in+neurologic+disease%5BTitle%5D)+AND+%22Neurotherapeutics%22%5BJournal%5D)">Caspases: therapeutic targets in neurologic disease.</a> Neurotherapeutics. 12 (1), 42-48 (2015).</li>
<li>Avrutsky, M. I., Troy, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Caspase-9%3A+a+multimodal+therapeutic+target+with+diverse+cellular+expression+in+human+disease%5BTitle%5D)+AND+%22Frontiers+in+Pharmacology%22%5BJournal%5D)">Caspase-9: a multimodal therapeutic target with diverse cellular expression in human disease.</a> Frontiers in Pharmacology. 12, 1728(2021).</li>
<li>Fuchs, Y., Steller, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Programmed+cell+death+in+animal+development+and+disease%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Programmed cell death in animal development and disease.</a> Cell. 147 (4), 742-758 (2011).</li>
<li>Troy, C. M., Akpan, N., Jean, Y. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regulation+of+caspases+in+the+nervous+system%3A+implications+for+functions+in+health+and+disease%5BTitle%5D)+AND+%22Progress+in+Molecular+Biology+and+Translational+Science%22%5BJournal%5D)">Regulation of caspases in the nervous system: implications for functions in health and disease.</a> Progress in Molecular Biology and Translational Science. 99, 265-305 (2011).</li>
<li>Avrutsky, M. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Endothelial+activation+of+caspase-9+promotes+neurovascular+injury+in+retinal+vein+occlusion%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">Endothelial activation of caspase-9 promotes neurovascular injury in retinal vein occlusion.</a> Nature Communications. 11 (1), 3173(2020).</li>
<li>Colon Ortiz, C., Potenski, A., Lawson, J. M., Smart, J., Troy, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optimization+of+the+retinal+vein+occlusion+mouse+model+to+limit+variability%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Optimization of the retinal vein occlusion mouse model to limit variability.</a> Journal of Visualized Experiments. (174), e62980(2021).</li>
<li>Tisch, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Caspase-8+modulates+physiological+and+pathological+angiogenesis+during+retina+development%5BTitle%5D)+AND+%22The+Journal+of+Clinical+Investigation%22%5BJournal%5D)">Caspase-8 modulates physiological and pathological angiogenesis during retina development.</a> The Journal of Clinical Investigation. 129 (12), 5092-5107 (2019).</li>
<li>Chi, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((HMGB1+promotes+the+activation+of+NLRP3+and+caspase-8+inflammasomes+via+NF-kappaB+pathway+in+acute+glaucoma%5BTitle%5D)+AND+%22Journal+of+Neuroinflammation%22%5BJournal%5D)">HMGB1 promotes the activation of NLRP3 and caspase-8 inflammasomes via NF-kappaB pathway in acute glaucoma.</a> Journal of Neuroinflammation. 12, 137(2015).</li>
<li>Thomas, C. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Caspase-2+mediates+site-specific+retinal+ganglion+cell+death+after+blunt+ocular+injury%5BTitle%5D)+AND+%22Investigative+Ophthalmology+%26+Visual+Science%22%5BJournal%5D)">Caspase-2 mediates site-specific retinal ganglion cell death after blunt ocular injury.</a> Investigative Ophthalmology & Visual Science. 59 (11), 4453-4462 (2018).</li>
<li>Akpan, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intranasal+delivery+of+caspase-9+inhibitor+reduces+caspase-6-dependent+axon%2Fneuron+loss+and+improves+neurological+function+after+stroke%5BTitle%5D)+AND+%22Journal+of+Neuroscience%22%5BJournal%5D)">Intranasal delivery of caspase-9 inhibitor reduces caspase-6-dependent axon/neuron loss and improves neurological function after stroke.</a> Journal of Neuroscience. 31 (24), 8894-8904 (2011).</li>
<li>London, A., Benhar, I., Schwartz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+retina+as+a+window+to+the+brain-from+eye+research+to+CNS+disorders%5BTitle%5D)+AND+%22Nature+Reviews+Neurology%22%5BJournal%5D)">The retina as a window to the brain-from eye research to CNS disorders.</a> Nature Reviews Neurology. 9 (1), 44-53 (2013).</li>
<li>Song, P., Xu, Y., Zha, M., Zhang, Y., Rudan, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Global+epidemiology+of+retinal+vein+occlusion%3A+a+systematic+review+and+meta-analysis+of+prevalence%2C+incidence%2C+and+risk+factors%5BTitle%5D)+AND+%22Journal+of+Global+Health%22%5BJournal%5D)">Global epidemiology of retinal vein occlusion: a systematic review and meta-analysis of prevalence, incidence, and risk factors.</a> Journal of Global Health. 9 (1), 010427(2019).</li>
<li>Akpan, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intranasal+delivery+of+caspase-9+inhibitor+reduces+caspase-6-dependent+axon%2Fneuron+loss+and+improves+neurological+function+after+stroke%5BTitle%5D)+AND+%22The+Journal+of+neuroscience%22%5BJournal%5D)">Intranasal delivery of caspase-9 inhibitor reduces caspase-6-dependent axon/neuron loss and improves neurological function after stroke.</a> The Journal of neuroscience. 31 (24), 8894-8904 (2011).</li>
<li>Gage, G. J., Kipke, D. R., Shain, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Whole+animal+perfusion+fixation+for+rodents%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Whole animal perfusion fixation for rodents.</a> Journal of Visualized Experiments. (65), e3564(2012).</li>
<li>McStay, G. P., Salvesen, G. S., Green, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Overlapping+cleavage+motif+selectivity+of+caspases%3A+implications+for+analysis+of+apoptotic+pathways%5BTitle%5D)+AND+%22Cell+Death+%26+Differentiation%22%5BJournal%5D)">Overlapping cleavage motif selectivity of caspases: implications for analysis of apoptotic pathways.</a> Cell Death & Differentiation. 15 (2), 322-331 (2007).</li>
<li>Kuida, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reduced+apoptosis+and+cytochrome+c-mediated+caspase+activation+in+mice+lacking+caspase+9%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Reduced apoptosis and cytochrome c-mediated caspase activation in mice lacking caspase 9.</a> Cell. 94 (3), 325-337 (1998).</li>
<li>Troy, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Death+in+the+balance%3A+alternative+participation+of+the+caspase-2+and+-9+pathways+in+neuronal+death+induced+by+nerve+growth+factor+deprivation%5BTitle%5D)+AND+%22Journal+of+Neuroscience%22%5BJournal%5D)">Death in the balance: alternative participation of the caspase-2 and -9 pathways in neuronal death induced by nerve growth factor deprivation.</a> Journal of Neuroscience. 21 (14), 5007-5016 (2001).</li>
</ol>

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 c...

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><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&amp;nbsp;</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 ...

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

Research

JoVE Journal

Neuroscience

1.4K 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

Lighting Up the Pathways to Caspase Activation Using Bimolecular Fluorescence Complementation

The caspase family of proteases play essential roles in apoptosis and innate immunity. Among these, a subgroup known as initiator caspases are the first to be activated in these pathways. This group includes caspase-2, -8, and -9, as well as the inflammatory caspases, caspase-1, -4, and -5. The initiator caspases are all activated by dimerization following recruitment to specific multiprotein complexes called activation platforms. Caspase Bimolecular Fluorescence Complementation (BiFC) is an imaging-based approach where split fluorescent proteins fused to initiator caspases are used to visualize the recruitment of initiator caspases to their activation platforms and the resulting induced proximity. This fluorescence provides a readout of one of the earliest steps required for initiator caspase activation. Using a number of different microscopy-based approaches, this technique can provide quantitative data on the efficiency of caspase activation on a population level as well as the kinetics of caspase activation and the size and number of caspase activating complexes on a per cell basis.

This protocol describes caspase Bimolecular Fluorescence Complementation (BiFC); an imaging-based method that can be used to visualize induced proximity of initiator caspases, which is the first step in their activation.

The caspase family of proteases play essential roles in apoptosis and innate immunity. Among these, a subgroup known as initiator caspases are the first ...

Biochemistry

Characterization of Molecular Mechanisms of In vivo UVR Induced Cataract

Cataract is the leading cause of blindness in the world 1. The World Health Organization defines cataract as a clouding of the lens of the eye which impedes the transfer of light. Cataract is a multi-factorial disease associated with diabetes, smoking, ultraviolet radiation (UVR), alcohol, ionizing radiation, steroids and hypertension. There is strong experimental 2-4 and epidemiological evidence 5,6 that UVR causes cataract. We developed an animal model for UVR B induced cataract in both anesthetized 7 and non-anesthetized animals 8.
The only cure for cataract is surgery but this treatment is not accessible to all. It has been estimated that a delay of onset of cataract for 10 years could reduce the need for cataract surgery by 50% 9. To delay the incidence of cataract, it is needed to understand the mechanisms of cataract formation and find effective prevention strategies. Among the mechanisms for cataract development, apoptosis plays a crucial role in initiation of cataract in humans and animals 10. Our focus has recently been apoptosis in the lens as the mechanism for cataract development 8,11,12. It is anticipated that a better understanding of the effect of UVR on the apoptosis pathway will provide possibilities for discovery of new pharmaceuticals to prevent cataract.
In this article, we describe how cataract can be experimentally induced by in vivo exposure to UVR-B. Further RT-PCR and immunohistochemistry are presented as tools to study molecular mechanisms of UVR-B induced cataract.

Characterization of Molecular Mechanisms of In vivo UVR Induced Cataract

Cataract is the leading cause of blindness in the world. Solar ultraviolet radiation (UVR) is the main risk factor for cataract development. An animal model of far UVR-B induced cataract was developed. In this article we describe methods for investigation of cataract formation: exposure to UVR, quantitative RT-PCR and immunohistochemistry.

Cataract is the leading cause of blindness in the world 1. The World Health Organization defines cataract as a clouding of the lens of the eye which ...

Medicine

Caspase-3 Activity in the Rat Amygdala Measured by Spectrofluorometry After Myocardial Infarction

Myocardial infarction (MI) has dramatic mid- and long-term consequences at the physiological and behavioral levels, but the mechanisms involved are still unclear. Our laboratory has developed a rat model of post-MI syndrome that displays impaired cardiac functions, neuronal loss in the limbic system, cognitive deficits and behavioral signs of depression. At the neuronal level, caspase-3 activation mediates post-MI apoptosis in different limbic regions, such as the amygdala &#8211; peaking at 3 days post-MI. Cognitive and behavioral impairments appear 2-3 weeks post-MI and these correlate statistically with measures of caspase-3 activity. The protocol described here is used to induce MI, collect amygdala tissue and measure caspase-3 activity using spectrofluorometry. To induce MI, the descending coronary artery is occluded for 40 min. The protocol for evaluation of caspase-3 activation starts 3 days after MI: the rats are sacrificed and the amygdala isolated rapidly from the brain. Samples are quickly frozen in liquid nitrogen and kept at -80 &#176;C until actual analysis. The technique performed to assess caspase-3 activation is based on cleavage of a substrate (DEVD-AMC) by caspase-3, which releases a fluorogenic compound that can be measured by spectrofluorometry. The methodology is quantitative and reproducible but the equipment required is expensive and the procedure for quantifying the samples is time-consuming. This technique can be applied to other tissues, such as the heart and kidneys. DEVD-AMC can be replaced by other substrates to measure the activity of other caspases.

Myocardial infarction (MI) is not only followed by impaired cardiac function but also by apoptosis in the amygdala, a brain region involved in the behavioral consequences of MI. This protocol describes how to induce MI, collect amygdala tissue and measure caspase-3 activity, a marker of apoptosis, therein.

Myocardial infarction (MI) has dramatic mid- and long-term consequences at the physiological and behavioral levels, but the mechanisms involved are still ...

Visualization of Inflammatory Caspases Induced Proximity in Human Monocyte-Derived Macrophages

Inflammatory caspases include caspase-1, -4, -5, -11, and -12 and belong to the subgroup of initiator caspases. Caspase-1 is required to ensure correct regulation of inflammatory signaling and is activated by proximity-induced dimerization following recruitment to inflammasomes. Caspase-1 is abundant in the monocytic cell lineage and induces maturation of the pro-inflammatory cytokines interleukin (IL)-1&#946; and IL-18 to active secreted molecules. The other inflammatory caspases, caspase-4 and -5 (and their murine homolog caspase-11) promote IL-1&#946; release by inducing pyroptosis. Caspase Bimolecular Fluorescence Complementation (BiFC) is a tool used to measure inflammatory caspase induced proximity as a readout of caspase activation. The caspase-1, -4, or -5 prodomain, which contains the region that binds to the inflammasome, is fused to non-fluorescent fragments of the yellow fluorescent protein Venus (Venus-N [VN] or Venus-C [VC]) that associate to reform the fluorescent Venus complex when the caspases undergo induced proximity. This protocol describes how to introduce these reporters into primary human monocyte-derived macrophages (MDM) using nucleofection, treat the cells to induce inflammatory caspase activation, and measure caspase activation using fluorescence and confocal microscopy. The advantage of this approach is that it can be used to identify the components, requirements, and localization of the inflammatory caspase activation complex in living cells. However, careful controls need to be considered to avoid compromising cell viability and behavior. This technique is a powerful tool for the analysis of dynamic caspase interactions at the inflammasome level as well as for the interrogation of the inflammatory signaling cascades in living MDM and monocytes derived from human blood samples.

This protocol describes the workflow to obtain monocytes-derived macrophages (MDM) from human blood samples, a simple method to efficiently introduce inflammatory caspase Bimolecular Fluorescence Complementation (BiFC) reporters into human MDM without compromising cell viability and behavior, and an imaging-based approach to measure inflammatory caspase activation in living cells.

Inflammatory caspases include caspase-1, -4, -5, -11, and -12 and belong to the subgroup of initiator caspases. Caspase-1 is required to ensure correct ...

Immunology and Infection

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

Evaluation of Caspase Activation to Assess Innate Immune Cell Death

Innate immunity provides the critical first line of defense in response to pathogens and sterile insults. A key mechanistic component of this response is the initiation of innate immune programmed cell death (PCD) to eliminate infected or damaged cells and propagate immune responses. However, excess PCD is associated with inflammation and pathology. Therefore, understanding the activation and regulation of PCD is a central aspect of characterizing innate immune responses and identifying new therapeutic targets across the disease spectrum.
This protocol provides methods for characterizing innate immune PCD activation by monitoring caspases, a family of cysteine-dependent proteases that are often associated with diverse PCD pathways, including&#160;apoptosis,&#160;pyroptosis, necroptosis, and PANoptosis. Initial reports characterized caspase-2, caspase-8, caspase-9, and caspase-10 as initiator caspases and caspase-3, caspase-6, and caspase-7 as effector caspases in apoptosis, while later studies found the inflammatory caspases, caspase-1, caspase-4, caspase-5, and caspase-11, drive pyroptosis. It is now known that there is extensive crosstalk between the caspases and other innate immune and&#160;cell death molecules across the previously defined PCD pathways, identifying a key knowledge gap in the mechanistic understanding of innate immunity and PCD and&#160;leading to the characterization of PANoptosis. PANoptosis is a unique innate immune inflammatory PCD pathway regulated by PANoptosome complexes, which integrate components, including caspases,&#160;from other cell death pathways.
Here, methods for assessing the activation of caspases in response to various stimuli are provided. These methods allow for the characterization of PCD pathways both in vitro and in vivo, as activated caspases undergo proteolytic cleavage that can be visualized by western blotting using optimal antibodies and blotting conditions. A protocol and western blotting workflow have been established that allow for the assessment of the activation of multiple caspases from the same cellular population, providing a comprehensive characterization of the PCD processes. This method can be applied across research areas in development, homeostasis, infection, inflammation, and cancer to evaluate PCD pathways throughout cellular processes in health and disease.

This protocol describes a comprehensive method for assessing caspase activation (caspase-1, caspase-3, caspase-7, caspase-8, caspase-9, and caspase-11) in response to both in vitro and in vivo (in mice) models of infection, sterile insults, and cancer to determine the initiation of cell death pathways, such as pyroptosis, apoptosis, necroptosis, and PANoptosis.

Innate immunity provides the critical first line of defense in response to pathogens and sterile insults. A key mechanistic component of this response is ...

Immunostaining and AI-Based Counting of RGCs in Mouse Whole-Mount Retina

Whole-Mount Immunostaining and Automatic Counting of Mouse Retinal Ganglion Cells

Glaucoma is a leading cause of blindness globally, characterized by a complex pathogenic mechanism that makes vision restoration challenging. Mice serve as valuable animal models for studying the pathogenesis and treatment of glaucoma due to their relatively homogenous genetic background and retinal ganglion cells (RGCs), which structurally resemble those in humans. Accurate assessment of RGC damage and treatment outcomes in mouse glaucoma models requires determining the RGC number across the entire retina. This protocol outlines a comprehensive method involving the isolation of the whole retina, labeling RGCs with specific antibodies, and rapid, accurate automatic counting of RGCs using an AI-based program. The streamlined approach allows for efficient and precise quantification of RGC numbers in mouse retinas, facilitating the evaluation of RGC degeneration and potential therapeutic interventions. By enabling researchers to assess the extent of RGC damage, this protocol contributes to a deeper understanding of glaucoma pathogenesis and aids in developing effective treatment strategies to manage and prevent vision loss.

Author Spotlight: Efficient Retinal Ganglion Cell Counting in Mouse Models of Glaucoma for Treatment Evaluation

This protocol describes the procedure for isolating the whole-mount mouse retina and performing immunostaining to label all retinal ganglion cells (RGCs). The process is followed by imaging and automatically counting RGCs using AI-based software, providing a simple, fast, and accurate method for quantifying RGCs in the entire mouse retina.

Glaucoma is a leading cause of blindness globally, characterized by a complex pathogenic mechanism that makes vision restoration challenging. Mice serve ...

Novel Retina Protection Assay for Diabetic Retinopathy Pathogenesis Study

An Assay to Detect Protection of the Retinal Vasculature from Diabetes-Related Death in Mice

Diabetic retinopathy (DR) is a complex and progressive ocular disease characterized by two distinct phases in its pathogenesis. The first phase involves the loss of protection from diabetes-induced damage to the retina, while the second phase centers on the accumulation of this damage. Traditional assays primarily focus on evaluating capillary degeneration, which is indicative of the severity of damage, essentially addressing the second phase of DR. However, they only indirectly provide insights into whether the protective mechanisms of the retinal vasculature have been compromised. To address this limitation, a novel approach was developed to directly assess the retina's protective mechanisms - specifically, its resilience against diabetes-induced insults like oxidative stress and cytokines. This protection assay, although initially designed for diabetic retinopathy, holds the potential for broader applications in both physiological and pathological contexts. In summary, understanding the pathogenesis of diabetic retinopathy involves recognizing the dual phases of protection loss and damage accumulation, with this innovative protection assay offering a valuable tool for research and potentially extending to other medical conditions.

Author Spotlight: Understanding Retinal Vessel Resilience and Disease Progression

A protection assay was developed to monitor the retinal vasculature's resilience to death from diabetes/diabetic retinopathy-related insults such as oxidative stress and cytokines.

Diabetic retinopathy (DR) is a complex and progressive ocular disease characterized by two distinct phases in its pathogenesis. The first phase involves ...

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. ...

Sampling of N-methyl-N-nitrosourea-induced Rat's Retina Damage and Multi-index Evaluation of Pathological Changes

Retinopathy can be observed in most ocular diseases and the late complications of diabetes mellitus. The specific pigment epithelial cells and optic cells&#39; damage and degeneration are the main features of retinopathy. Many&#160;traditional medicines have shown substantial clinical efficacy in treating retinopathy. How to obtain a retina quickly and completely is a key step in traditional medicine research for the treatment of retinopathy. In this study, we aim to provide a standardized and exercisable procedure for sampling of N-methyl-N-nitrosourea (MNU)-induced rat&#39;s retina damage and multi-index evaluation of pathological changes. Rats were injected intraperitoneally with 60 mg/kg MNU once to induce retina damage, and retina samples were obtained after 7 days. Additionally, we performed hematoxylin-eosin staining to assess retinal pathological&#160;changes.&#160;Determination of the apoptosis rate and apoptosis protein by TUNEL and Western blot. These standardized protocols for retinal sampling and evaluation of pathological changes are helpful in promoting the exploration of the mechanism of retinopathy and the discovery of novel and effective traditional herbs.

This protocol describes a procedure for rat retinal sampling and utilizing hematoxylin-eosin staining, TUNEL assay, and Western blot to detect pathological changes and apoptosis in the retina after intraperitoneal injection of N-Methyl-N-Nitrosourea.

Retinopathy can be observed in most ocular diseases and the late complications of diabetes mellitus. The specific pigment epithelial cells and optic ...

Quantification of Immunostained Caspase-9 in Retinal Tissue

Summary

Explore More Videos

Lighting Up the Pathways to Caspase Activation Using Bimolecular Fluorescence Complementation

Characterization of Molecular Mechanisms of In vivo UVR Induced Cataract

Caspase-3 Activity in the Rat Amygdala Measured by Spectrofluorometry After Myocardial Infarction

Visualization of Inflammatory Caspases Induced Proximity in Human Monocyte-Derived Macrophages

Optimized Protocol for Retinal Wholemount Preparation for Imaging and Immunohistochemistry

Evaluation of Caspase Activation to Assess Innate Immune Cell Death

Whole-Mount Immunostaining and Automatic Counting of Mouse Retinal Ganglion Cells

An Assay to Detect Protection of the Retinal Vasculature from Diabetes-Related Death in Mice

Cryosectioning and Immunostaining of Mouse Retina

Sampling of N-methyl-N-nitrosourea-induced Rat's Retina Damage and Multi-index Evaluation of Pathological Changes