The overall goal of this procedure is to selectively label retinal ganglion cells in the whole mount preparation via an optic nerve stump injection of a calcium indicator dye. This is accomplished by first injecting a very high affinity calcium indicator dye, such as flow four into the optic nerve stump. Next, the retina is isolated from the eye cup and divided into quadrants to mount onto a glass slide In a recording chamber set up to mimic physiological conditions, the contributions of voltage gated calcium channels to the calcium signal.
In retinal ganglion cells can be measured in depolarized cells using channel blockers. Ultimately, the results can provide semi-quantitative measurements of the contribution of the voltage educated calcium channels to a signal such as a high potassium evoked calcium signal. The main advantage of this technique over existing methods, such as electroporation and bulk loading, is the ability to selectively label retinal ganglion cells and their axons in a whole mount preparation Store, dissected rad eyes and hibernate a aims medium or similar solution.
Now, if you intend to perform calcium imaging experiments, backfill the retinal ganglion cells with flow four. Inject 0.5 microliters of 40 millimolar flow four penta potassium salt, reconstituted in water into the optic nerve, stump approximately one millimeter posterior to the eyeball. Transfer the eyeball to mammalian ringers.
Bubbled with 95%oxygen, 5%carbon dioxide, or an hour at room temperature. Keep the preparation in the dark. Dissect the eyes under a microscope with controlled light intensity.
Using a razor blade gently make a small incision in the front of the eyeball and remove the cornea. Then remove the lens from the inner retinal surface using forceps. Hold the sclera firmly.
When removing the vitreous with the other forceps, gently peel the vitreous base toward the center of the retina. The resulting preparation called the I cup can be processed for immunohistochemical whole mount preparation, calcium imaging preparation, or prepared for transverse retinal sections to make an immunohistochemical whole mount preparation. First, make four cuts to the retina so it lays flat.
Place the retina on the glass cover slide with the ganglion cell layer facing down. Second, add a drop of solution to the retina and cover it with cellulose filter paper if needed, flatten the retina with a brush or forceps. Third, when the retina is completely flat and detach to the filter paper, place it back into the solution.
To process the preparation first, transfer it to 4%paraform aldehyde for 10 to 15 minutes. Then wash the retinas three times in 0.1 molar phosphate buffer at pH 7.4 for 30 minutes per wash for a total of 90 minutes. Next, incubate the retinas in 500 microliters of blocking solution overnight at four degrees Celsius.
This solution is 5%normal goat serum or donkey serum in phosphate buffer with 0.3%tritton and 0.1%sodium azide over the next five to seven days, incubate the retinas in the primary antibody solution at four degrees Celsius. The makeup of this solution must be arrived at empirically. Wash off the primary antibody solution just as the fixative was washed off.
Then incubate the retinas in secondary antibody at a concentration of one to 1000 overnight at four degrees Celsius. Remove the secondary antibody with three more washes and phosphate buffer. Then mount the retinas for visualization and seal with nail polish.
These slides must be stored in the dark at four degrees Celsius. Begin with making a calcium imaging whole mount preparation from the I cup in ringers. First, cut the retinal tissue into quadrants.
Place one quadrant flat on the recording chamber with the ganglion layer facing up, and thus the photoreceptor layer facing down. Carefully dry the surrounding area with a Kim wipe and gently place the greased harp on the tissue. Now load the retinal preparation on the rig and finish setting up the eight channel gravity driven superfusion system.
If the mounted retina moves after the harp is placed, carefully remove and clean the harp. Dry the area surrounding the retina and remount the harp super. Fuse the recording chamber with mammalian ringer solution and keep the solution bubbling continuously with 95%oxygen, 5%carbon dioxide.
First, as a control without drugs, perform two paired high potassium pulses from three millimolar to 60 millimolar for 33 seconds. Depolarizing, the RGC should activate the voltage gated calcium channels if the preparation is working. Now proceed with the experiments.
Nifedipine and L type voltage gated calcium channel blocker is commonly used to assess the contribution of L type voltage gated calcium channels to the depolarization evoked calcium signal cobalt. A non-specific calcium channel blocker can be used to nons selectively block voltage gated calcium channels. Scan the prep every five seconds to visualize and acquire images of the fluorescent rgc.
Use as low a laser power as possible. Excitation should be set to 488 nanometers and emission should be collected with a 505 nanometer LP filter. Using the described protocols, immuno labeling was performed against the ganglion cell protein R-B-P-M-S, which is an RNA binding protein with multiple splicing flow.Four.
Staining was indeed limited to ganglion cells based on the antibody specification. Next, the contribution of the voltage gated calcium channels to the calcium signal in the R GCs was studied using the fluorescent intensity values acquired from the rgc. At baseline levels, RGC and RGC axons can be seen labeled with flow four.
In order to acquire fluorescent intensity values, ROIs were placed on the ganglion cell, somata and or axons. After applying high potassium to the whole mount preparation, the fluorescent signal of the RG C'S increased. This was followed by a subsequent reduction in fluorescence intensity values in the presence of cobalt during the second high potassium pulse.
After watching this video, you should have a good understanding of how to selectively load retinal ganglion cells with a synthetic calcium indicator die in a whole mount preparation.
