To begin, assemble a dissecting microscope, two forceps with fine tips, scissors, a one milliliter syringe needle, and filter paper on a working platform. Next, using elbow scissors, nucleate the eyes from an anesthetized mouse. Place the eyes into a glass dish containing 4%paraformaldehyde and 0.2%picric acid in 0.1 molar phosphate buffer.
On the dissecting microscope, using a one milliliter syringe, punch a hole at the corneal limbus and cut off the anterior segment with scissors. Use forceps to remove the lens from the inner retinal surface. Now using two forceps, carefully peel the sclera until the retina is completely isolated from the eye cup.
Subsequently, cut the retina into four pieces. After overnight fixation and 4%paraformaldehyde solution, wash the retinal tissues in 0.01 molar PBS six times for 10 minutes each. Incubate the retinal tissue in 1%sodium borohydride in 0.01 molar PBS for 30 minutes.
Again, wash the retinal sections in 0.01 molar PBS at least six times. Then, remove the vitreous with filter paper. And using a double-edged razor blade, slice the retina into small sections between 100 and 300 micrometers.
Wash the retinal stripes in 0.01 molar PBS six times for 10 minutes each before incubating them with Reagent A and Reagent B from the ABC kit for two days. Next, wash the retinal stripes in 0.05 molar tris buffer three times for 10 minutes each. Pre-incubate the retinal stripes with 5%Reagent 1 and 5%Reagent 3 from the diaminobenzene, or DAB kit, in distilled water for one hour at room temperature.
To stain the retinal stripes with DAB, add the same volume of solution from three tubes in the DAB kit in sequence. Under the dissection microscope, observe the staining condition. Wash the retinal strips with 0.05 molar tris buffer three times for 10 minutes each.
Finally, wash the strips in 0.01 molar PBS six times for 10 minutes each. In control experiments, cone pedicles with two ribbons and rod spherical with one ribbon showed no immunoreactivity in the absence of primary antibodies. Protein kinase C alpha identified rod bipolar cells in the retina.
The DAB standing showcases the presence of protein kinase C alpha and bipolar cell dendrites, forming synapses with rod and cone terminals in the outer plexiform layer. Additionally, the DAB reaction product aids in identifying rod bipolar cell terminals and axonal processes in the inner plexiform layer. Substance P immunoreactivity amacrine cells were shown to be presynaptic to substance P negative amacrine cells.
Furthermore, substance P immunoreactivity amacrine cells were post-synaptic to bipolar terminals in the sublayer three and sublayer five levels of the inner plexiform layer, respectively.
