Double Immunostaining via antigen retrieval

1. Fix the tissue for 4 hours @ RT in 4% MEMFA (Made in water)
2. Wash the tissue with PBS 3x10' @ RT on a shaker
3. Put in 30% sucrose and shake O/N at 4˚C (cold room)
4. Embed in OCT and freeze on Dry ice and transfer the frozen block to -20˚C freezer until sectioning
5. Cut sections as desired, let them dry for at least 1 hour @ RT. Contour with Dako pen
6. Wash the slides 3x10' in PBS-Tween 20 (0.3% Tween20) in plastic container
7. Block for 1h @ RT in PBS, 1% BSA, 0.3% Triton X100 (blocking buffer)
8. Add RFP/Cherry/GFP/ or any other antibody (MBP 1:200, b3TUB 1:200, MHC, MEF2c etc)that does not require antigen retrieval diluted in blocking buffer and dispense 250-300µl per slide. Incubate O/N in the cold room.
9. Wash 5x10' with PBS-Tween 20 at RT in plastic container
10. Add (250-300µl per slide) secondary antibody (Alexa's) diluted in blocking buffer (1:200 dilution) and incubate for 1h @ RT
11. Mount in 50% Glycerol-­‐PBS. Put coverslips on the slides but don't put nail Polish. Image the slides if possible to be on the safe side and to make sure the staining worked.

If you don't want to image then just go from step 10 to 13.