Investigating Retinal Circuits and Molecular Localization by Pre-Embedding Immunoelectron Microscopy

This protocol outlines the detailed steps of pre-embedding immunoelectron microscopy, with a focus on exploring synaptic circuits and protein localization in the retina.

In our research, we focus on retinal physiology and the disease. They need to investigate the cellular composition and the specific neurotransmitters within the retinal neural circuit. The overarching goal is to explore synaptic connections and the cellular localization of neurotransmitters throughout the entire pathway.

The biggest challenge of immunoelectron microscope technology is difficult in achieving a balance between structure, integrity, and positive signals. Through pre-embedding immunoelectron microscopy, we provide for the first time, immunochemically verified ultrastructural evidence for the coalescent synapse. And the review and explicit distribution, novel organic cell features, and synaptic organization or SPIR in the mouse retina.

All findings suggest that the cross-talk between cone and the rod pathway is much more extensive than previously assumed. This neurochemically identified ultrastructural results review that on and off signaling may interconnect through chemical synapse in the retina's OPL. 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, enucleate 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 in 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 diaminobenzidine or DAB kit and 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 staining showcases the presence of protein kinase C alpha in rod 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 amicron cells were shown to be presynaptic to substance P negative amicron cells. Furthermore, substance P immunoreactivity amicron cells were postsynaptic to bipolar terminals in the sub layer three and sub layer five levels of the inner plexiform layer respectively.