I am trying to do double IHC-p/IF on mouse lung tissue. For the 1st antigen, Fas, I have a polyclonal rabbit-anti-mouse Fas primary antibody and a goat-anti-rabbit AF488 secondary antibody.
For the 2nd antigen, vimentin, I have a polyclonal goat-anti-mouse vimentin primary antibody and a donkey-anti-goat AF546 secondary antibody.
I have successfully done single stains with each of them separately, which roughly follows this protocol:
- Rehydrate from xylene to ethanol to ddH2O
- Antigen retrieval with Dako citrate buffer at 90 degrees C in hot water bath for 60 min
- For vimentin stain, I add in a permeabilization step here with 0.3% Triton X-100 in TBS-T at RT for 30 min
- Rinse 2x 5 min with TBS-T + 0.025% Triton X-100
- Block with TBS-T + 3% BSA + 1% serum (of secondary antibody host species) for 60 min
- Apply primary antibody diluted in blocking buffer and incubate overnight at 4 degrees C
- Rinse 2x 5 min with TBS-T + 0.025% Triton X-100
- Apply secondary antibody diluted in blocking buffer and incubate for 90 min at RT, keeping sections covered from now on to avoid photobleaching
- Rinse 3x 5 min with TBS-T
- Apply room temperature 0.2% Sudan Black B dissolved in 70% EtOH to sections to reduce autofluorescence, incubate for 15 min
- Rinse 2x 5 min with DI H2O
- Apply DAPI nuclear stain to sections and incubate for 15 min
- Rinse 1x 5 min with DI H2O
- Coverslip with glycerol diluted to 80% with TBS-T, seal with clear nail polish
- Allow approx 30 min to dry and then image
Again, single stains following this protocol have worked on their own. When I try to do double staining, I basically added in both primary antibodies during same overnight incubation, and then added in both secondary antibodies in same 90 min incubation, so the total time to do the protocol is the same.
Fas shows up great ... vimentin does not show up at all. Any ideas?
Thank you!