Name: two peeling methods for the isolation of photoreceptor cell compartments in the mouse retina for protein analysis
Uploaded: 2021-12-07T13:00:00
Description: Watch this Scientific Journal Video about Two Peeling Methods for the Isolation of Photoreceptor Cell Compartments in the Mouse Retina for Protein Analysis at JoVE.com

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

All experiments were performed according to the local institutional guidelines of the committee on research animal care from the University of Southern California (USC).
1. Live cell retinal peeling method
<ol>
	<li>Preparation of Ames&#39; buffers, peeling papers, and dissection dish
	<ol>
		<li>Using blunt tip iris scissors (or equivalent scissor type), cut the cellulose filter paper (Grade 413) into rectangles measuring approximately 5 mm x 2.5 mm. Store the cuttings for ...

<ol>
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L., Dawson, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic+light+intensity+threshold+for+retinal+damage+in+albino+rats+raised+under+6+lx.">Cyclic light intensity threshold for retinal damage in albino rats raised under 6 lx.</a> Experimental Eye Research. 44 (5), 643-661 (1987).</li><li>Brann, M. R., Cohen, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diurnal+expression+of+transducin+mRNA+and+translocation+of+transducin+in+rods+of+rat+retina.">Diurnal expression of transducin mRNA and translocation of transducin in rods of rat retina.</a> Science. 235 (4788), 585-587 (1987).</li><li>Philp, N. 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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+isolation+of+retinal+outer+segment+fragments.">The isolation of retinal outer segment fragments.</a> The Journal of Cell Biology. 27 (3), 459-473 (1965).</li><li>Tsang, S. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+for+the+target+enzyme+in+deactivation+of+photoreceptor+G+protein+in+vivo.">Role for the target enzyme in deactivation of photoreceptor G protein in vivo.</a> Science. 282 (5386), 117-121 (1998).</li><li>Walston, S. T., Chow, R. H., Weiland, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patch+clamp+recordings+of+retinal+bipolar+cells+in+response+to+extracellular+electrical+stimulation+in+wholemount+mouse+retina.">Patch clamp recordings of retinal bipolar cells in response to extracellular electrical stimulation in wholemount mouse retina.</a> Annual International Conference of the IEEE Engineering in Medicine and Biology Society. 2015, 3363-3366 (2015).</li><li>Guido, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+to+obtain+retinal+cell+preparations+highly+enriched+in+specific+cell+types.+Suitability+for+lipid+metabolism+studies.">A simple method to obtain retinal cell preparations highly enriched in specific cell types. Suitability for lipid metabolism studies.</a> Brain Research. Brain Research Protocols. 4 (2), 147-155 (1999).</li><li>Hayashi, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorylation+by+cyclin-dependent+protein+kinase+5+of+the+regulatory+subunit+of+retinal+cGMP+phosphodiesterase.+II.+Its+role+in+the+turnoff+of+phosphodiesterase+in+vivo.">Phosphorylation by cyclin-dependent protein kinase 5 of the regulatory subunit of retinal cGMP phosphodiesterase. II. Its role in the turnoff of phosphodiesterase in vivo.</a> The Journal of Biological Chemistry. 275 (42), 32958-32965 (2000).</li><li>Wolbring, G., Cook, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+purification+and+characterization+of+protein+kinase+C+from+bovine+retinal+rod+outer+segments.">Rapid purification and characterization of protein kinase C from bovine retinal rod outer segments.</a> European Journal of Biochemistry. 201 (3), 601-606 (1991).</li><li>Williams, D. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+protein+kinase+C+in+photoreceptor+outer+segments.">Characterization of protein kinase C in photoreceptor outer segments.</a> Journal of Neurochemistry. 69 (4), 1693-1702 (1997).</li><li>Watson, A. J., Aragay, A. M., Slepak, V. Z., Simon, M. 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Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+GTPase+activating+factor+for+transducin+in+rod+photoreceptors+is+the+complex+between+RGS9+and+type+5+G+protein+beta+subunit.">The GTPase activating factor for transducin in rod photoreceptors is the complex between RGS9 and type 5 G protein beta subunit.</a> Proceedings of the National Academy of Sciences of the United States of America. 96 (5), 1947-1952 (1999).</li><li>Chen, J., Shi, G., Concepcion, F. A., Xie, G., Oprian, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+rhodopsin/arrestin+complex+leads+to+retinal+degeneration+in+a+transgenic+mouse+model+of+autosomal+dominant+retinitis+pigmentosa.">Stable rhodopsin/arrestin complex leads to retinal degeneration in a transgenic mouse model of autosomal dominant retinitis pigmentosa.</a> The Journal of Neuroscience. 26 (46), 11929-11937 (2006).</li><li>Chen, C. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abnormal+photoresponses+and+light-induced+apoptosis+in+rods+lacking+rhodopsin+kinase.">Abnormal photoresponses and light-induced apoptosis in rods lacking rhodopsin kinase.</a> Proceedings of the National Academy of Sciences of the United States of America. 96 (7), 3718-3722 (1999).</li><li>Zhang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mistrafficking+of+prenylated+proteins+causes+retinitis+pigmentosa+2.">Mistrafficking of prenylated proteins causes retinitis pigmentosa 2.</a> FASEB Journal. 29 (3), 932-942 (2015).</li><li>Weiss, E. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Species-specific+differences+in+expression+of+G-protein-coupled+receptor+kinase+(GRK)+7+and+GRK1+in+mammalian+cone+photoreceptor+cells:+implications+for+cone+cell+phototransduction.">Species-specific differences in expression of G-protein-coupled receptor kinase (GRK) 7 and GRK1 in mammalian cone photoreceptor cells: implications for cone cell phototransduction.</a> The Journal of Neuroscience. 21 (23), 9175-9184 (2001).</li><li>Zhao, X., Huang, J., Khani, S. C., Palczewski, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+forms+of+human+rhodopsin+kinase+(GRK1).">Molecular forms of human rhodopsin kinase (GRK1).</a> The Journal of Biological Chemistry. 273 (9), 5124-5131 (1998).</li><li>Newton, A. C., Williams, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+protein+kinase+C+in+the+phosphorylation+of+rhodopsin.">Involvement of protein kinase C in the phosphorylation of rhodopsin.</a> The Journal of Biological Chemistry. 266 (27), 17725-17728 (1991).</li><li>Pinzon-Guzman, C., Zhang, S. S., Barnstable, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+protein+kinase+C+isoforms+are+required+for+rod+photoreceptor+differentiation.">Specific protein kinase C isoforms are required for rod photoreceptor differentiation.</a> The Journal of Neuroscience. 31 (50), 18606-18617 (2011).</li><li>Sokal, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+protein+kinase+C+isozymes+responsible+for+the+phosphorylation+of+photoreceptor-specific+RGS9-1+at+Ser475.">Identification of protein kinase C isozymes responsible for the phosphorylation of photoreceptor-specific RGS9-1 at Ser475.</a> The Journal of Biological Chemistry. 278 (10), 8316-8325 (2003).</li><li>Rose, K., Walston, S. 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Many retinal diseases affect rod photoreceptor cells, leading to rod death and, ultimately, complete vision loss37. A significant portion of the genetic and mechanistic origins of human retinal degeneration have been successfully recapitulated in numerous mouse models over the years. In that context, the ability to easily and selectively separate individual rod subcellular compartments from the small mouse retina would greatly enhance our understanding of the localized biochemical and molecular un...

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

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

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

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

The present strategies were developed to provide relatively rapid and simple methods to isolate and analyze proteins among specific rod subcellular compartments for western blot analysis. We applied two sequential peeling techniques (Figure 1 and Figure 2) followed by immunoblotting to demonstrate that these methods could reliably be used to detect the known distribution of rod transducin (GNAT1) and arrestin (ARR1) in both dark- and light-adapted animals. To va...

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

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

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

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

The two peeling techniques demonstrated here isolate individual rod subcellular compartments, and can help reveal the important physiologic processes occurring in each specialized compartment in healthy and diseased rods. Isolating different rod cell compartments from the mouse retina can be challenging. These simple techniques use inexpensive and commonplace lab materials to reliably isolate rod subcellular compartments for protein analysis.To begin, place a piece of filter paper into a Petri dish filled with Ames HEPES buffer bubbled with 100%oxygen. Then, using a wide-bore transfer pipette, transfer one retinal rectangle dissected out from a two to three-months-old C57 Black 6J mouse. Orient the partitioned retina concave up and ensure that the photoreceptors are facing down toward the bottom of the dish.Then, using tweezers, lightly grasp the sides of the halved retina and move it on top of the filter paper. Once the retina is centered, move the filter paper upward so the halved retina touches the filter paper to create rod outer segment, or ROS to filter paper adhesion. Carefully lift the filter paper with the attached retina out of the Ames HEPES buffer.Place the bottom side of the filter paper on a paper towel and dab two to three times to dry. Add a drop of Ames HEPES onto the side of the filter paper with the retina and place the filter paper on the paper towel again to dry. Next, place the filter paper with the retina back into the Petri dish, and using tweezers, gently push all edges of the halved retina up and away from the filter paper on all sides.Delicately peel the retina away from the filter paper, touching only the extreme perimeter of the retina to preserve its structural integrity. Then, lift the filter paper from the Petri dish and remove excess liquid on a paper towel, ensuring that the side in contact with the retina does not contact the paper towel. Place the filter paper with the partial ROS layer into a labeled tube on ice, and keep the peeled retina submerged in Ames HEPES buffer.Repeat the peeling process for this halved retina approximately seven to eight times to remove the entire ROS layer. After each peel, place the filter paper into the same tube to combine all ROS isolated from one halved retina. Keep the tube containing all filter paper peels of the isolated ROS on ice for immediate use.Using tweezers, transfer the leftover peeled retina into an appropriately-labeled tube and keep the tube on ice for immediate use. To prepare the retinal sample for lyophilization, place a piece of filter paper and transfer a halved retina into a Petri dish filled with cold Ringer's buffer. Next, using tweezers, carefully flip the retina so it is oriented concave up and move it to rest on top of the filter paper.Lift the filter paper by the edges upward so the halved piece of retina touches the filter paper. Continue to lift the filter paper out of Ringer's solution. Then, place the bottom side of the filter paper on a paper towel and carefully dab two to three times to remove excess liquid.Next, pick up the filter paper with tweezers and add a drop of cold Ringer's onto the side of the filter paper with the retina. Again, place the filter paper on the paper towel to remove excess liquid. Store each adhered retina and filter paper sample in a Petri dish filled with cold Ringer's until ready to freeze all samples.Then, using tweezers, lift each sample out of the cold Ringer's buffer, ensuring that the tweezers contact only the filter paper edges, avoiding the halved retina. Place the bottom side of each filter paper on a paper towel to remove excess liquid. Then, add a drop of cold PBS onto side of the filter paper where the retina is adhered and dab the filter paper on the paper towel again.Place all filter paper pieces into the Petri dish, and using lint-free tissue paper, wick away any excess liquid surrounding the filter paper. Then, tightly wrap the entire dish with aluminum foil and ensure that the edges of the aluminum foil are secured and smoothly pressed into the bottom of the Petri dish. Puncture a handful of 0.1 to 0.2-millimeter holes into the aluminum foil lid.Next, using metal tongs, gradually lower the aluminum foil-covered Petri dish into liquid nitrogen. Keep the samples in liquid nitrogen until ready to lyophilize. For lyophilization, place the aluminum foil-covered Petri dish into a freeze drying flask and attach it to the lyophilizer machine following the manufacturer's protocol.Lyophilize the retinae for 30 minutes. After lyophilization, collect the rod outer and inner segment by carefully laying a small piece of tape on top of the lyophilized retina and applying slight pressure with the tweezers to ensure it bonds with the photoreceptor layer. Slowly peel the tape away and ensure that both the ROS and RIS have adhered to the tape.A thin, white film will be present at the fractured surface. This is the RIS. To separate this RIS, place another piece of tape and push the tape down on the fractured surface.Then, place the piece of tape with only the orange-pink layer into a microcentrifuge tube labeled as ROS, and pieces of tape with the thin, white layer into another tube labeled RIS. Next, using tape, collect the remaining retinal tissue located on the filter paper by peeling off the thick, white layer. Then, place this isolate into a tube labeled OIS.If using the same day, store the isolated layers from the lyophilized retinae at room temperature. In dark-adapted animals, the distribution of GNAT1 and ARR1 closely matched their known dark-state distributions, where GNAT1 signal was visibly strongest in the ROS isolation, and the ARR1 signal was more robust in the ROS isolation. Quantification of the GNAT1 and ARR1 signals showed significant differences between dark-light conditions for GNAT1 and ARR1 in ROS and ROS samples.In both dark and light-adapted samples, G beat 5S, cytochrome C, and actin signals are excluded from the ROS samples, demonstrating an absence of contamination from other cellular layers. Additionally, G beta 5L signal was clearly visible in the ROS samples. Scanning electron microscopy of the surfaces of intact and peeled lyophilized retinae showed that, before peeling with adhesive tape, the surface of intact lyophilized retina closely matched the characteristic cylindrical ROS.After the initial tape peel, the ROS and RIS appeared to be entirely removed, as evidenced by the uniform nuclear layer present on the surface of the leftover, peeled, lyophilized retina. Subsequent tape peels removed RIS from the free surface of the peeled layer. This technique also demonstrated that GRK1 immunoreactivity was most intense in ROS samples in light-exposed retinae, and absent in OIS samples.Conversely, ROS and RIS samples displayed a faint signal for PKC alpha, which is commonly not detected in ROS or RIS by immunofluorescence staining. Finally, suboptimal tape peeling could yield slightly contaminated samples. The RIS sample was contaminated with some OIS sample, producing both G beta 5L and G beta 5S bands, and a higher PKC alpha signal.Care must be taken when lyophilizing the retina. If an improper lyophilization technique is used, the retina may melt, making isolating different rod subcellular layers by tape impossible. Using this tape peeling method in conjunction with qRT-PCR has allowed for the separation of photoreceptor transcripts from the rest of the retina.

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