This article details a protocol for immunofluorescence staining of rat coronal tissue cryosections to visualize microglia and neurons. The method includes blocking non-specific binding sites, incubating with primary and secondary antibodies, and imaging the sections under a fluorescence microscope.
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, should be from the species the secondary antibody is raised in. In this case, donkey serum was used.
Ensure the blocking solution extends to the edge of the slide and completely covers the tissue to avoid uneven staining. Place the slides into a humidity chamber to incubate at room temperature, for one hour. During incubation, prepare the primary antibodies. Dilute the IBA1 antibody, 1 to 5,000 and the pan neuronal antibody, 1 to 500 into a mixture of 1% donkey serum in PBS.
Remove blocking solution, and add 300 microliters of the antibody solution, which contains both antibodies of interest. Place all the slides into a humidity chamber, and incubate them overnight at 4 degrees Celsius. The following morning, wash the slides three times in PBS for five minutes each.
Prepare the fluorescent secondary antibody mixture, by diluting both of them 1 to 250, in 1% donkey serum in PBS. Add 300 microliters of the mixture onto each slide. And place the slides into a light, tight humidity chamber for 60 minutes at room temperature. Then wash the slides three times in PBS for five minutes each. And finally, rinse them in double distilled water. Protect the slides from light while washing in PBS.
Add a few drops of an aqueous mounting medium, that helps to preserve fluorescence signal intensity to each slide. Then coverslip the slide, and remove any remaining air bubbles using a cotton-tipped applicator. To seal the mounting media onto the slides and prevent the sections drying out, coat the edges with nail polish. The slides are left to dry flat for one hour in a light protected box, before being stored in a light, tight container, wrapped in foil at 4 degrees centigrade.
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