This procedure uses mouse retinal tissue to demonstrate how to stabilize delicate neural tissue to preserve structural integrity. During several steps of immunohistochemical staining, first nucleate the eye, remove the cornea and separate the retina from the sclera. Make incisions into the retinal eye cup to flatten the tissue.
Then attach the retina to a ring enforced membrane using a suction transfer without direct manipulation to preserve the integrity of the tissue. Now transfer the ring to a multi-well plate for staining. Our results show that this technique can be used effectively in live tissue and provides a superior substrate for the imaging of synaptic Proteins compared to existing metals of handling dissected retina.
These techniques allows for the transfer of delicate tissue with minimal physical manipulation. This will reduce the chance of distortion and damage. We first had this idea when we needed to record from flat mounted tissue in RD one mouse, a model of retinal degeneration, and this mouse retina are extremely thin and fragile due to extensive cell loss caused by the mutation Chief.
A postdoc in our lab will join me in demonstrating the procedure. First, nucleate the eyes of an anesthetized mouse and transfer the eyes into oxygenated heaps buffered ringer solution. To remove the cornea, hold the eye at the limbus with forceps, and then cut a circular path along the ora serrata.
Pull out the lens with forceps for bolus injections and electrophysiological recordings. Use blended edge forceps to remove as much vitreous as possible. Now stabilize the eye cup by holding the sclera firmly with forceps.
Position a second set of forceps between the retina and the eye cup. Move the forceps slowly around the circumference. Cut the optic nerve between the retina and the eye cup, and remove the eye cup with scissors.
Make incisions from the retinal periphery all the way down to the optic nerve. Alternatively, to use the entire retina, make incisions from the periphery halfway to the optic nerve to shorten the holder. Remove the excessive part with a lathe.
Then polish down rough surfaces with fine sandpaper. Remove the feet from underneath the ring. Cut off the narrow tip of a plastic pipette.
Then transfer the tissue with some solution to the membrane. Gradually remove the solution while spreading the retina and unfold the tissue. Now attach the retina to the membrane with the ganglion cells facing up.
Cut off the holding tip of a one milliliter syringe and position it directly underneath the tissue. Pull back on the plunger to a volume of about 0.3 milliliters to physically en mesh the tissue within the membrane. Pull some boro silicate capillary pipettes using a resistance of approximately one mega.
Then gently break the tip of each patch pipette. Next, add 0.3 microliters of is selectin stock stain solution to 10 microliters of the heaps buffered ringer solution. Now place the membrane into the recording chamber perfused with warm oxygenated heaps buffered ringer solution.
Position the pipette containing dye against the inner limiting membrane using a microm manipulator. Further advance the pipette to penetrate the inner limiting membrane. Then inject the staining solution into the single or multiple targeted locations.
Now allow 10 minutes for the tissue to recover from the injection while viewing it under epi fluorescence illumination. If desired, transfer the insert to a glass bottom culture dish, placing it retina side down. Add one drop of heap solution to keep the specimen moist.
Also, place three spots of vacuum grease around the perimeter of the plastic ring to firmly secure the insert to the culture dish. Transfer the dish to the microscope stage after fixation, submerge the membrane into the sucrose gradient solutions. Excise the membrane from the plastic insert ring.
Remove as much solution as possible with Kim wipes and place it onto param with the retina facing up. Transfer the paraform with the tissue to the cryostat chamber and add a drop of OCT medium. Use the quick freezer device to accelerate the freezing.
Detach the frozen sample and flip it over. Now cut off the excess frozen OCT. Leave approximately two millimeters around the sample on three sides and millimeters from the side, which will be attached to the holder.
Cover the exposed retinal side with a drop of OCT leaving the attached area free. Freeze the membrane containing the retinal tissue. Place a drop of OCT onto a cold holder ring.
Immediately insert the frozen sample into the drop. Cut 10 to 20 micron sections at negative 19 degrees Celsius and position them on warm polylysine adhesion slides. Place the slides with the attached sections on a 41 degree Celsius heating platform for two to three minutes in the dark.
Then store the samples at negative 20 degrees Celsius. This experiment illustrates a fast and simple characterization of retinal vasculature as an elaborate network of blood vessels that span across multiple retinal layers. By combining a bolus loading of iso selectin with sulfa rumine immersion labeling, living tissue can be visualized and scanned almost immediately in the retina, blood vessels can be labeled from the superficial to the deep layer.
In contrast to relatively well diffusible, SRH is selectin does not permeate well across the inner limiting membrane. There are several advantages of fixation with carbimide over conventional fixation with para formaldehyde when labeling synaptic proteins, for example, after a short period of fixation with carbimide immunofluorescent staining for the synaptic marker. PSD 95 in the IPL had a bright punctate appearance consistent with distinguishing the individual synapses.
In contrast, when using conventional paraform aldehyde fixative, identification of the synaptic components is less clear. Similar results were obtained in the retinal cryostat sections. After watching this video, you should have a good understanding how to mount a delicate tissue on this membrane and how to apply cyto chemical staining to the preparation one master.
It'll take you 15 minutes to attach the retina to the filter.
