1. Choose desired slides (paraformaldyhyde-fixed, 10 micron thick crysections).
2. Leave the chosen slides at room temperature just until the frost melts.
3. Rinse the slides 3 times in 1x TBST for 8 minutes each.
4. Block with 5% milk powder in 1x TBST for 30 minutes at room temperature. Optional: 1-2% BSA can be added to TBST.
5. Incubate overnight at 4oC with primary antibodies in TBST plus 5% milk.
6. Rinse for 10 seconds, 1x TBST. Then, rinse 3 times in 1x TBST for 8 minutes each.
7. Add secondary antibodies in TBST for 1 hour at room temperature in the dark.
8. Rinse 3 times in 1x TBS for 8 minutes each.
9. Add Fluorescent Mounting Medium, coverslip, and examine under a fluorescent microscope.
10. Lastly, seal the edges of covered-slip slides with a clear nail polish. This helps slides retain fluorescence longer during storage.
TBST = TBS + 0.1% Triton