Immunostaining of Fluorescently Labeled Astrocytes in a Mouse Brain Tissue Section

Take a fixed mouse brain tissue section containing a sparse population of astrocytes expressing a fluorescent protein.

Treat the free-floating section with a detergent-containing buffer to permeabilize the cells.

Incubate the section in a blocking buffer to prevent non-specific antibody binding.

Treat the section with primary antibodies and incubate to allow the antibodies to bind to the fluorescent proteins in the astrocytes.

Wash off any excess primary antibodies using buffer.

Next, incubate the section with fluorophore-tagged secondary antibodies that bind to the primary antibodies.

Wash off unbound secondary antibodies with a buffer.

Transfer the section to a buffer-deionized water mixture to remove detergent residues.

Place the section on a slide containing buffer-deionized water. Remove excess liquid and add mounting media. Then, position a coverslip and secure it.

Finally, use confocal microscopy to visualize the labeled fluorescent protein-expressing astrocytes within the section.

To begin, prepare a fresh solution of TBST by adding 1 milliliter of 10% Triton X to a 50-milliliter tube and filling the tube to 50 milliliters with TBS. Prepare 2 milliliters of blocking and ant