Immunohistochemical Staining of Rat Coronal Tissue Cryosections for Microglia and Neurons

Take a slide containing fixed and permeabilized rat coronal tissue cryosections.

Outline the sections with a hydrophobic pen to create a controlled staining environment.

Transfer the sections into a humidity chamber to prevent drying of reagents and tissue during subsequent steps.

Treat the sections with serum proteins to block non-specific binding sites.

Rinse with buffer to remove excess serum proteins.

Incubate with primary antibodies targeting specific proteins on microglia and neurons.

Wash with buffer to remove unbound primary antibodies.

Incubate with fluorophore-conjugated secondary antibodies that bind to the primary antibodies.

Wash with buffer to remove unbound secondary antibodies. Then, rinse with double-distilled water.

Apply aqueous mounting media to protect the fluorescent signal, place a coverslip, and seal it.

Image the sections under a fluorescence microscope to visualize immunolabeled microglia and neurons.

Dry the edges of the slides, and make a liquid repellent border around the edge of the slide with a pap pen, to ensure an even covering of the liquid and reduce edge effects. Next, block the sections by adding 300 microliters of serum in PBS to each slide. The serum, s