Double Immunostaining via antigen retrieval: Difference between revisions
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# Wash 5x10' with PBS-Tween 20 at RT in plastic container | # Wash 5x10' with PBS-Tween 20 at RT in plastic container | ||
# Add (250-300µl per slide) secondary antibody (Alexa's) diluted in blocking buffer (1:200 dilution) and incubate for 1h @ RT | # Add (250-300µl per slide) secondary antibody (Alexa's) diluted in blocking buffer (1:200 dilution) and incubate for 1h @ RT | ||
# 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. | |||
Revision as of 13:50, 20 February 2013
- Fix the tissue for 4 hours @ RT in 4% MEMFA (Made in water)
- Wash the tissue with PBS 3x10' @ RT on a shaker
- Put in 30% sucrose and shake O/N at 4˚C (cold room)
- Embed in OCT and freeze on Dry ice and transfer the frozen block to -20˚C freezer until sectioning
- Cut sections as desired, let them dry for at least 1 hour @ RT. Contour with Dako pen
- Wash the slides 3x10' in PBS-Tween 20 (0.3% Tween20) in plastic container
- Block for 1h @ RT in PBS, 1% BSA, 0.3% Triton X100 (blocking buffer)
- 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.
- Wash 5x10' with PBS-Tween 20 at RT in plastic container
- Add (250-300µl per slide) secondary antibody (Alexa's) diluted in blocking buffer (1:200 dilution) and incubate for 1h @ RT
- 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.