I am currently performing IHC for human tissue microarrays but all my tissues, including negative control where I did not add any primary antibody, stained positive. I block peroxidase with H2O2 band lock unspecific binding with 1% casein.
I am thinking of trying 5% BSA-PBS, 5% human albumin and 5% FCS.
Any suggestions?
Try blocking with a 5%BSA/5% serum of the secondary antibody species, in PBS or TBS, whichever you use. For example, if you are using a donkey anti-rabbit secondary antibody (assuming you are using a secondary antibody), then use 5% BSA/5% donkey serum. You want to block secondary antibody binding.
Make sure you use the serum that your secondary was raised in. For example. If you are using a goat anti rabbit secondary , then I would use goat serum to block non-specific binding of my secondary antibody.
Hello Flourina.
Rehaydrate tissues, incubate in plenty of 0.26% NH4Cl solution to block active aldehyde groups for 30 min, changing once at 15 min. This aldehyde groups bind any nitrogen compounds, therefore block them first. Reduce antibody concentrations and repeated washes for 20 min each with plenty of buffer containing 0.05% (v/v) Tween 20.
Best wishes.
I typically use 3% Peroxide block for 15 min and throughly wash it with water and then move to PBST. For non-specific binding I use 5% normal goat serum with a pinch of BSA and incubate between 30 min to 1 hour. Also make sure your Xylenes and Alcohol washes are clean. Sometimes leftover dyes can interfere with the tissue
I would suggest that you first check whether your problem is caused by endogenous peroxidases or alternatively by unspecific binding of your reagents (such as detection antibody, biotin, etc).
I am assuming that you are using a kit that relies on a biotin/conjugated detection antibody and some sort of streptavidin-conjuagated peroxidase?
To narrow down the source of your problem, you should:
1- Start by just adding the substrate onto your slides (no Abs, bio/strep, etc). Do you still get a signal? If yes, you probably have a problem with endogenous peroxidases after all. Maybe your H2O2 has gone off.
2- Next possibility is that your tissue contains endogenous biotin or streptavidin. Again, you can leave these out and check if you get a signal. If this is the case you can buy dedicated blocking agents for bio/strep.
3- Finally, your primary or secondary antibody might be the problem. As you mentioned, you have already tested for the first, so try different concentrations of the secondary and see how the signal turns out. To optimize this stage you can also play with conventional blocking reagents such as BSA, or detergents such as Tween20.
Bear in mind that staining quality can also be affected by the tissue fixation and antigen retrieval steps. Was your sample frozen or paraffinized?
Good luck!
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