1. Cut less than 40 μm cryostat free floating sections.
2. Antigen repair with Citrate Buffer if necessary (98℃ treat 5 minutes and wait for to RT).
3. Rinse sections with TBS, 10 min.
4. Incubate sections in 0.3% Triton-x-100 in TBS for 60 min at RT.
5. Incubate sections with Blocking Solution (5% goat serum, 3% BSA in TBS, pH = 7.2) for 1.5 hours at RT.
6. Aspirate Blocking Solution. Incubate sections with primary antibodies (diluted in Blocking Solution) overnight at 4℃.
7. Balanced at RT for about 30 minutes.
8. Rinse with TBS three times for 10 minutes each.
9. Incubate sections with secondary antibodies (diluted in TBS) in RT about 2 hours and protect from light.
10. Rinse with TBS 10 min.
11. Incubate in DAPI (1:1000 diluted in TBS) for 10 minutes.
12. Rinse with TBS three times for 10 minutes each.
13. Suspend brain slices in TBS (with little TBST) to hold sections to slides. (If your sections are attached to slides immediately after slicing, ignore this step)
14. Coverslip the slides with aqueous mounting medium containing anti-fade. Store at RT overnight to dry and then transfer to refrigerator.