Double Immunostaining via antigen retrieval: Difference between revisions

Revision as of 10:31, 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

@@ Line 3: / Line 3: @@
 # 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