Tissue
Immunofluorescence protocol

1. Cryostat section 6 microns and put onto glass slides.
2. Keep slides at –20 C until ready for use.
3. Do not let slides dry up, keep in liquid at all times until end.
4. Rehydrate with 2 washes of 1xPBS.
5. 3 washes .5% BSA.
6. BLOCK with 2% BSA for 45 minutes.
7. 5 washes of .5% BSA.
8. Primary antibody for 60 minutes. (made in .5% BSA, vortex, spin down)
9. 5 washes of BSA.
10. Secondary antibody for 60 minutes. (made in .5% BSA, vortex, spin down)
11. 5 washes of .5% BSA.
12. 5 washes of PBS.
13. Hoescht stain for 30 seconds.
14. 3 washes with PBS.
15. Cover slip with gelvatol and refrigerate.
16. Slides must dry overnight and be stored at 4 C in the dark.

Do not let tissue sections dry up, keep in liquid at all times until gelvatol step.

Spin down antibodies for 5 minutes at maximum rpm.

2% BSA = 2g BSA per 100mL 1xPBS

0.5% BSA = .5g BSA per 100 mL 1xPBS (for all washes and antibodies)