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 antibody solutions for each brain by combining goat serum and TBST in a 15-milliliter tube.

Next, label a 24-well plate to place different samples in different rows, in different solutions, in different columns. Then, add 1 milliliter of TBST to the first three columns labeled as 'Wash 1', 'Wash 2', and 'Wash 3', and add 1 milliliter of blocking solution to the fourth column.

Prepare a glass pick by melting the end of a 5.75-inches Pasteur pipette into a small hook using a Bunsen burner. Then transfer tissue sections from the 12-well plate into the 'Wash 1' column of the 24-well plate using the pick. Then wash the sections for 10 minutes each in Wash 1, 2, and 3 wells by using the glass pick to transfer the sections from one well to the next, followed by incubating the sections for one hour in the blocking solution.

Next, incubate the sections in primary antibody for two to three nights at 4 degrees Celsius while shaking. After primary antibody incubation, aspirate the day one TBST from the wash wells. Then add 1 milliliter of new TBST to each wash well, and move the sections into the first wash well.

Next, incubate sections in secondary antibody for three hours at room temperature. After secondary antibody incubation, aspirate the day two TBST from the wash wells. Then add 1 milliliter of new TBST to each wash well and move the sections into the first wash well.

During the final wash, take the mounting media out from 4 degrees Celsius and allow it to warm to room temperature. Then prepare a 2 to 1 mixture of TBS in deionized water and add it to a Petri dish. Next, prepare a microscope slide by adding 800 microliters of 2 to 1 mixture of TBS in deionized water to the surface.

Transfer the sections from the 'Wash 3' well to the Petri dish. Then, using a fine paintbrush, transfer the sections one at a time from the Petri dish into the liquid on the slide. Next, carefully arrange the sections so that they are flat on the slide using the paintbrush. Carefully remove excess liquid from the slide with a P1000 pipette, followed by vacuum aspiration.

Once all excess liquid is removed from the slide, use a transfer pipette to immediately add one drop of mounting media to each section and gently lay a coverslip over the slide. Allow mounting media to spread for a few minutes, and then remove any excess mounting media that comes out from under the coverslip by vacuum aspiration. Afterward, seal all four edges of the coverslip with clear nail polish.