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

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

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

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RetNet, the Retinal Information Network Available from: <a target="_blank" href="https://sph.uth.edu/retnet">https://sph.uth.edu/retnet</a> (2014)</li><li>Mohand-Said, S., Hicks, D., Dreyfus, H., Sahel, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+transplantation+of+rods+delays+cone+loss+in+a+retinitis+pigmentosa+model.">Selective transplantation of rods delays cone loss in a retinitis pigmentosa model.</a> Arch Ophthalmol. 118 (6), 807-811 (2000).</li><li>MacLaren, R. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+repair+by+transplantation+of+photoreceptor+precursors.">Retinal repair by transplantation of photoreceptor precursors.</a> Nature. 444 (7116), 203-207 (2006).</li><li>Yang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+photoreceptor+and+total+neural+retina+preserves+cone+function+in+P23H+rhodopsin+transgenic+rat.">Transplantation of photoreceptor and total neural retina preserves cone function in P23H rhodopsin transgenic rat.</a> PLoS One. 5 (10), (2010).</li><li>Mohand-Said, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+retina+releases+a+diffusible+factor+stimulating+cone+survival+in+the+retinal+degeneration+mouse.">Normal retina releases a diffusible factor stimulating cone survival in the retinal degeneration mouse.</a> Proc Natl Acad Sci USA. 95 (14), 8357-8362 (1998).</li><li>Fintz, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Partial+characterization+of+retina-derived+cone+neuroprotection+in+two+culture+models+of+photoreceptor+degeneration.">Partial characterization of retina-derived cone neuroprotection in two culture models of photoreceptor degeneration.</a> Invest Ophthalmol Vis Sci. 44 (2), 818-825 (2003).</li><li>Cronin, T., Leveillard, T., Sahel, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+degenerations:+from+cell+signaling+to+cell+therapy;+pre-clinical+and+clinical+issues.">Retinal degenerations: from cell signaling to cell therapy; pre-clinical and clinical issues.</a> Curr Gene Ther. 7 (2), 121-129 (2007).</li><li>Wright, A. 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H., Holmgren, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thioredoxin+and+related+molecules--from+biology+to+health+and+disease.">Thioredoxin and related molecules--from biology to health and disease.</a> Antioxid Redox Signal. 9 (1), 25-47 (2007).</li><li>Fridlich, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+thioredoxin-like+protein+rod-derived+cone+viability+factor+(RdCVFL)+interacts+with+TAU+and+inhibits+its+phosphorylation+in+the+retina.">The thioredoxin-like protein rod-derived cone viability factor (RdCVFL) interacts with TAU and inhibits its phosphorylation in the retina.</a> Mol Cell Proteomics. 8 (6), 1206-1218 (2009).</li><li>Yang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+cone+rescue+by+RdCVF+protein+in+a+dominant+model+of+retinitis+pigmentosa.">Functional cone rescue by RdCVF protein in a dominant model of retinitis pigmentosa.</a> Mol Ther. 17 (5), 787-795 (2009).</li><li>Bennett, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+for+delivery+of+rod-derived+cone+viability+factor.">Strategies for delivery of rod-derived cone viability factor.</a> Retina. 25 (8 Suppl), S47 (2005).</li><li>Komaromy, A. 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The authors have nothing to disclose.

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

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

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			<td height="21" style="height:21px;width:319px;"><a name="RANGE!A1:D28">Name of Material / Equipment</a></td>
			<td style="width:167px;">Company</td>
			<td style="width:131px;">Catalog Number</td>
			<td style="width:368px;">Comments/Description</td>
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			<td height="21" style="height:21px;">Cytidine 5&#8242;-diphosphocholin*</td>
			<td>Sigma-Aldrich</td>
			<td>C0256</td>
			<td>4.7 &#181;M Cytidine 5&#8242;-diphosphocholin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cytidine 5&#8242;-diphosphoethanolamine*</td>
			<td>Sigma-Aldrich</td>
			<td>C0456</td>
			<td>2.7 &#181;M Cytidine 5&#8242;-diphosphoethanolamine</td>
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		<tr height="21">
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			<td>Sigma-Aldrich</td>
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		<tr height="21">
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			<td>T6397</td>
			<td>0.03 &#181;M Triiodo-L-thyronine</td>
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		<tr height="21">
			<td height="21" style="height:21px;">96-wells plate</td>
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			<td>655-095</td>
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			<td>Leica</td>
			<td>MZ-75</td>
			<td></td>
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		<tr height="26">
			<td height="26" style="height:26px;">CO2 independent (CO2-i)</td>
			<td>Life Technologies</td>
			<td>18045054</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">DMEM</td>
			<td>Life Technologies</td>
			<td>41966029</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Forceps n&#176;5 Dumont</td>
			<td>Bionic</td>
			<td>11254-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gelatin from porcin skin type A</td>
			<td>Sigma-Aldrich</td>
			<td>G2500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">GeneChip&#160;</td>
			<td>Affymetrix</td>
			<td>U74v2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gentamicin solution</td>
			<td>Life Technologies</td>
			<td>15710049</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hydrocortison*</td>
			<td>Sigma-Aldrich</td>
			<td>H0888</td>
			<td>0.55 &#181;M hydrocortison</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Insulin* (I)</td>
			<td>Sigma-Aldrich</td>
			<td>I1884 (ITS)</td>
			<td>0.86 &#181;M insulin (I)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Papain</td>
			<td>Worthington-biochem</td>
			<td>WOLS03124</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Poly-D-Lysine</td>
			<td>Sigma-Aldrich</td>
			<td>P-6407</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Progesterone*</td>
			<td>Sigma-Aldrich</td>
			<td>P7556&#160;</td>
			<td>2.0 &#181;M progesterone</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Prostaglandin*</td>
			<td>Sigma-Aldrich</td>
			<td>P5172</td>
			<td>0.28 &#181;M prostaglandin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Putrescine*</td>
			<td>Sigma-Aldrich</td>
			<td>P5780</td>
			<td>&#160;182 &#181;M putrescine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scalpel Albion</td>
			<td>EMS</td>
			<td>72000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scissor&#160;</td>
			<td>Moria</td>
			<td>15396-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Pyruvate*</td>
			<td>Sigma-Aldrich</td>
			<td>S8636</td>
			<td>1 mM sodium pyruvate</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">Sodium Selenite* (S)</td>
			<td>Sigma-Aldrich</td>
			<td>I1884 (ITS)</td>
			<td>0.29 &#181;M Na2SeO3&#160;&#160; (S)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Taurine*</td>
			<td>Sigma-Aldrich</td>
			<td>T8691</td>
			<td>3 mM taurine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Transferrin* (T)</td>
			<td>Sigma-Aldrich</td>
			<td>I1884 (ITS)</td>
			<td>0.07 &#181;M transferrin (T)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vibratome apparatus</td>
			<td>Leica</td>
			<td>VT1000-S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">* Supplements</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
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vibratome sectioning mouse retina to prepare photoreceptor cultures

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

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

Watch this Scientific Journal Video about Vibratome Sectioning Mouse Retina to Prepare Photoreceptor Cultures at JoVE.com

Vibratome Sectioning Mouse Retina to Prepare Photoreceptor Cultures

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

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

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

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JoVE Journal

Neuroscience

16.6K Views.  UMR_S 968, Institut de la Vision. Neural retina of a mouse aged 8 days is on top of a 4% gelatin block. After isolation of the photoreceptor layer (200 µm) by vibratome, the photoreceptors are seeded after mechanical and enzymatic dissociation for culture. The photoreceptor layer can be used for molecular, biochemical analyses or transplantation.

Video: Vibratome Sectioning Mouse Retina to Prepare Photoreceptor Cultures

Vibratome Sectioning for Enhanced Preservation of the Cytoarchitecture of the Mammalian Organ of Corti

The mammalian organ of Corti is a highly ordered cellular mosaic of mechanosensory hair and nonsensory supporting cells (reviewed in 1,2).Visualization of this cellular mosaic often requires that the organ of Corti is cross-sectioned. In particular, the nonsensory pillar and Deiters' cells, whose nuclei are located basally with respect to the hair cells, cannot be visualized without cross-sectioning the organ of Corti. However, the delicate cytoarchitecture of the mammalian organ of Corti, including the fine cytoplasmic processes of the pillar and Deiters' cells, is difficult to preserve by routine histological procedures such as paraffin and cryo-sectioning, which are compatible with standard immunohistochemical staining techniques.
Here I describe a simple and robust procedure consisting of vibratome sectioning of the cochlea, immunohistochemical staining of these vibratome sections in whole mount, followed by confocal microscopy. This procedure has been used widely for immunhistochemical analysis of multiple organs, including the mouse limb bud, zebrafish gut, liver, pancreas, and heart (see 3-6 for selected examples). In addition, this procedure was sucessful for both imaging and quantitificaton of pillar cell number in mutant and control organs of Corti in both embryos and adult mice 7. This method, however, is currently not widely used to examine the mammalian organ of Corti. The potential for this procedure to both provide enhanced preservation of the fine cytoarchitecture of the adult organ of Corti and allow for quantification of various cell types is described.

A simple procedure of vibratome sectioning the organ of Corti, followed by immunohistochemistry and confocal microscopy is described. This procedure allows for improved preservation of the fine cytoarchitecture of the mammalian organ of Corti, and consequently allows for accurate quantification of cell types.

The mammalian organ of Corti is a highly ordered cellular mosaic of mechanosensory hair and nonsensory supporting cells (reviewed in 1,2).Visualization of ...

In vivo Electroporation of Developing Mouse Retina

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

In vivo Electroporation of Developing Mouse Retina

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

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

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

Angiogenesis is the complex process of new blood vessel formation defined by the sprouting of new blood vessels from a pre-existing vessel network. Angiogenesis plays a key role not only in normal development of organs and tissues, but also in many diseases in which blood vessel formation is dysregulated, such as cancer, blindness and ischemic diseases. In adult life, blood vessels are generally quiescent so angiogenesis is an important target for novel drug development to try and regulate new vessel formation specifically in disease. In order to better understand angiogenesis and to develop appropriate strategies to regulate it, models are required that accurately reflect the different biological steps that are involved. The mouse neonatal retina provides an excellent model of angiogenesis because arteries, veins and capillaries develop to form a vascular plexus during the first week after birth. This model also has the advantage of having a two-dimensional (2D) structure making analysis straightforward compared with the complex 3D anatomy of other vascular networks. By analyzing the retinal vascular plexus at different times after birth, it is possible to observe the various stages of angiogenesis under the microscope. This article demonstrates a straightforward procedure for analyzing the vasculature of a mouse retina using fluorescent staining with isolectin and vascular specific antibodies.

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

The neonatal murine retina provides a well characterized physiological model of angiogenesis, which permits investigations of the roles of different genes or drugs that modulate angiogenesis in an in vivo context. Immunofluorescent staining to accurately visualize the vascular plexus is pivotal to the success of these types of studies.

Angiogenesis  is the complex process of new blood vessel formation defined by the sprouting  of new blood vessels from a pre-existing vessel network. ...

Assessment of Vascular Regeneration in the CNS Using the Mouse Retina

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

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

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

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

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

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

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

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

Cone-Enriched Cultures from the Retina of Chicken Embryos to Study Rod to Cone Cellular Interactions

Human daytime vision relies on the function of cone photoreceptors at the center of the retina, the fovea. Patients suffering from the most prevalent form of inherited retinal degeneration, retinitis pigmentosa, lose night vision because of mutation driven loss of rod photoreceptors, a phenomenon followed by a progressive loss of function and death of cones leading to blindness. Geneticists have identified many genes with mutations causing this disease, but the first mutations identified questioned the mechanisms of secondary cone degeneration and how a dominant mutation in the rhodopsin gene encoding for the visual pigment expressed exclusively in rods can trigger cone degeneration.
This result of transplantations in a genetic model of the disease led to the concept of cell interactions between rods and cones and of non-cell autonomous degeneration of cones in all genetic forms of retinitis pigmentosa.
Cones comprise 5% of all photoreceptors in humans and only 3% in the mouse, so their study is difficult in these species, but cones outnumber rods in bird species. We have adapted 96-well plates to culture retinal precursors from the retina of chicken embryos at stage 29 of their development. In these primary cultures, cones represent 80% of the cells after in vitro differentiation. The cells degenerate over a period of one week in the absence of serum. Here, we describe the methods and its standardization.
This cone-enriched culture system was used to identify the epithelium-derived cone viability factor (EdCVF) by high content screening of a rat retinal pigmented epithelium normalized cDNA library. Recombinant EdCVF prevents the degeneration of the cones.

We describe a method to obtain primary cultures of cone photoreceptors from the retina of chicken embryos and its use for high content screening.

Human daytime vision relies on the function of cone photoreceptors at the center of the retina, the fovea. Patients suffering from the most prevalent form ...

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

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

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

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

In Vivo Vascular Injury Readouts in Mouse Retina to Promote Reproducibility

Advancements in ophthalmic imaging tools offer an unprecedented level of access to researchers working with animal models of neurovascular injury. To properly leverage this greater translatability, there is a need to devise reproducible methods of drawing quantitative data from these images. Optical coherence tomography (OCT) imaging can resolve retinal histology at micrometer resolution and reveal functional differences in vascular blood flow. Here, we delineate noninvasive vascular readouts that we use to characterize pathological damage post vascular insult in an optimized mouse model of retinal vein occlusion (RVO). These readouts include live imaging analysis of retinal morphology, disorganization of retinal inner layers (DRIL) measure of capillary ischemia, and fluorescein angiography measures of retinal edema and vascular density. These techniques correspond directly to those used to examine patients with retinal disease in the clinic. Standardizing these methods enables direct and reproducible comparison of animal models with clinical phenotypes of ophthalmic disease, increasing the translational power of vascular injury models.

In Vivo Vascular Injury Readouts in Mouse Retina to Promote Reproducibility

Here, we present three data analysis protocols for fluorescein angiography (FA) and optical coherence tomography (OCT) images in the study of Retinal Vein Occlusion (RVO).

Advancements in ophthalmic imaging tools offer an unprecedented level of access to researchers working with animal models of neurovascular injury. To ...