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.