Hello!
I have tried to optimise my IHC protocol, but I still have positive cells in secondary antibody control sample (see picture attached) and now I'm thinking will anything help with that. I'm permeabilising tissues with TBS + 0.2% Triton for 10 min, blocking with 10% BSA, 5% goat serum and 5% FBS for 1h, primary antibody incubation in diluted block overnight at 4C and then block with 3% H2O2 in TBS for 30 min, sec antibody incubation in diluted block 30 min. DAB, hematoxylin, dehydration and finish with xylene. Between each step there is of course washes with TBS + 0.1% Tween.
Since I still have this kind of staining in sec ab control, then it is quite hard to determine lower signal from this background signal. I have seen that some use 3% H2O2 + methanol to block peroxidases, could this help? Any other ideas? Our sec ab are new and primaries are suitable for IHC. I'm trying to detect Ki67, CD206, CD86 and IFN-g in mouse tumor tissues.
Thank you!
Anni
Why don't you just switch to fluorescence if the DAB process causing too much non-specific signal? You can also use different channels and detect multiple targets in a single sample. Also, it looks like your tumor tissue has a particularly strong endogenous peroxidase level which can be useful for you since you can see the tumor cells infiltration. Is it lymphatic or gland tissue originated tumor cells? Methanol has little effect on endogenous peroxidases, where H2O2 is an excellent quenching agent, try to expose your samples for 30 minutes with 3% H2O2.
https://www.researchgate.net/post/Any_advice_on_how_to_improve_the_quality_of_immunohistochemistry_fluorescent_staining
If all control slide have that type of expression, means there have expression of the protein. You can try to do more wash your slide after staining.
I think permeabilization time can be increased up to 30 minutes at room temperature. You can wash the slides more too.
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