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.
