Difference between revisions of "Simple"
Revision as of 03:07, 27 June 2006
1. Choose desired slides (paraformaldyhyde-fixed, 10 micron thick crysections).
2. Leave the chosen slides at room temperature just until the frost melts.
6. Rinse the slides 3 times in 1x TBST for 8 minutes each.
7. Block with 5% milk powder in 1x TBST for 30 minutes at room temperature. Optional: 1-2% BSA can be added to TBST.
8. Incubate overnight at 4oC with primary antibodies in TBST plus 5% milk.
9. Rinse for 10 seconds, 1x TBST. Then, rinse 3 times in 1x TBST for 8 minutes each.
10. Add secondary antibodies in TBST for 1 hour at room temperature in the dark.
11. Rinse 3 times in 1x TBS for 8 minutes each.
12. Add Fluorescent Mounting Medium, coverslip, and examine under a fluorescent microscope.
13. Lastly, seal the edges of covered-slip slides with a clear nail polish. This helps slides retain fluorescence longer during storage.