Vibratome Sectioning Mouse Retina to Prepare Photoreceptor Cultures

The overall goal of this procedure is to isolate the mouse photoreceptor layer. For culturing, the first step is to obtain a complete flat retina and to seal it onto a gelatin block. Next, the retinal layers are successfully cut in order to isolate the photoreceptor layer.

The final step is to seed purified photoreceptor cells in culture. Ultimately, the purity of the photoreceptor layer is confirmed by microscopy and immunofluorescence, followed by rod derived cone viability factor testing. The main advantage of this technique over existing methods like laser micro dissection, is to confidently is isolate the photoreceptor layer.

The implications of this technology extend toward therapy of inherited retinal degeneration. Since it is very convenient to study photoreceptor biology, we had originally developed this method to study the cellular interaction between rod and cold photoreceptors. The transplantation of layer photoreceptor into the eye of the early one mar led to the identification of all derived con viability factors.

Visual demonstration of these methods is critical as the opportunity of flat mountain retina is difficult to perform because it is tricky. Before isolating the retina, prepare the vibrato. Start with dishes of 20%gelatin solution taken out of storage at four degrees Celsius.

Cut the gelatin with a scalpel, then flip the gelatin slice and stick it on the black support disc of the vibrator using a drop of super glue. Next, break a razor blade into two halves and insert one half into the vibrator. Then insert the black support disc onto the vibrator apparatus and turn the black knob to the right.

Now, secure the holding receptacle to the head of the vibrato and cover the gelatin block with CO2 independent medium. Switch on the vibrato to test out the setup. Cut three 100 micron slices until the slicing is smooth and flat slices are produced.

After harvesting the eyes from a mouse and other preparations, isolate the retina. First, use an 18 gauge needle to make a hole at the level of the eye limb. Then to remove the ciliary body, insert straight scissors through the hole and into the eye globe, and carefully cut the sclera below the iris, and then along the perimeter of the ocular globe.

Next, keep the posterior of the eye with the retina contiguous to the sclera and remove the cornea and the lens. With two fine grips, remove the vitreous, which is attached to the retina without damaging the retina so the retina can be laid flat. Make four radial cuts in it.

Now upturn the ocular globe and peel it like an orange. Next, very carefully with fine straight scissors. Detach the retina from the sclera and retinal pigmented epithelium.

It should stay attached at the level of the optic nerve using fine grips, detach the remaining vitreous starting at the periphery and moving towards the center of the retina. With all the vitreous completely removed, the retina will flatten. Now transfer the retina to a 35 millimeter diameter Petri dish.

The process of sealing the flat retina in the gelatin block is delicate and fundamentally the most challenging aspect to sectioning the tissue. Firstly, remove 20 to 30 milliliters of medium from the vibram tank. Secondly, transfer the retina to a glass slide using a plastic wide bore pipette and apply a drop of CO2 independent medium to the slide.

Thirdly, transfer the retina to a gelatin slice with a photoreceptor facing down. Fourthly, attach the retina to the gelatin block. Gently inject warm 4%gelatin on one side of the block between the retina and the gelatin block and expel gelatin onto the other side.

Lastly, aspirate the gelatin with a glass pipette and wait seven to 10 minutes while the retina is sealed into the block. Now add CO2 independent medium back to the tank, and immerse the block and blade for sectioning starting from the top of the gelatin block. Cut 100 micron serial sections until the blade reaches the retina.

Then using a very slow speed, like one or two cut 100 to 120 micron sections of the retina's inner layer. Depending on the application, this layer may be discarded or stored in liquid nitrogen, keeping the speed slow at the interface between the inner and outer layer. Take 15 micron sections.

Examine these sections under a microscope for the presence of blood vessels. When there are none, the outer retina has been reached through the outer layer. Slowly cut 200 micron sections.

Transfer these slices to a 35 millimeter dish with three milliliters of cold, CO2 independent medium kept on ice. Proceed with collecting all the photoreceptor layer sections from all of the retinas to be sectioned to this dish. Begin with transferring the sliced photoreceptors to a 37 degree Celsius incubator for 10 minutes.

Then using forceps, gently separate each photoreceptor layer from the gelatin and transfer them to a new dish filled with three milliliters of ringer solution at room temperature. Once all the photoreceptors have been isolated from the gelatin, combine two units of papain with 25 microliters of activator solution in a sterile five milliliter polypropylene tube and incubated for 30 minutes. To activate the papain during this incubation, cut the photoreceptor layer slices into two millimeter square pieces and not much smaller.

Transfer these pieces into a five milliliter tube with 1.5 milliliters of ringer solution. Then aspirate the ringers and replace it for a second wash. Wait for all the slices to settle by gravity, sedimentation, and then remove all remaining ringer solution from the tube.

Next, add 475 microliters of ringer solution to the tube containing the activated papain and mix. Transfer this mixture to the tube containing the slices and incubate them for 20 minutes. After 20 minutes, stop the papain digestion by adding one milliliter of 10%FCS in DMEM.

Then add 25 units of D Ns.One to digest the DNA from photoreceptor cells that have died carefully homogenize the suspension with pipetting to palate the photoreceptor cells. Spin the tube at 115 Gs for six minutes at room temperature, discard the supernatant and resuspend the cells in supplemented DMEM using a one milliliter pipette. Repeat this, spin and resus suspension.Once.

Now from the harvested eye, one to 1.5 million cells should be ready to seed. Seed them at 100, 000 cells per square centimeter and culture them for five days before downstream applications Using the described methods, cultures of photoreceptor cells were made from wild type mice at postnatal day eight. Without FCS, the cells were fixed and stained for anti row or an earlier marker anti sag.

Fewer cells were positive for anti row since SAG precedes ROE expression. Photoreceptors were also prepared from 35 day old mice to examine expression of mRNA, of RO sag and the cone transducing GA two. These genes express most prominently in the photoreceptors compared to the whole brain.

G NAT two is specifically expressed by cones. Protein expression of RO and g. NAT two in the photoreceptors was examined by comparing the photoreceptor layer cells with the inner retinal layer cells.

This was done with wild type and with RD mutant mice. The mutant mice showed no expression of either protein at day 35. After watching this video, you should have a good understanding of how to obtain a proper purified photoreceptor layer.

This technique can be done in about five to six hours for four to eight retinas if it is performed Properly following this procedure. Also, methods like western blood can be performed in order to answer additional question like the protein expression during the degeneration of photoreceptors After its development. This technique paved the way for researcher in the field of retinal biology to study the transcript, the proteome and the metabolism of relevant animal models.