Hi
I am working with a very specific antibody but it gives me a lot of aspecific signal on my tissue at the preferred dilution. The specific staining is nuclear but I see a faint signal in the surrounding tissue or in the interstitial tissue. I tried the avidin/biotin method and it gave me a brighter signal but not necessarily decreased the background. I was wondering if some of you have experience with the TSA method or even better have compared both methods. Any rationale to use one over the other?
Thanks,
Hi Florian, in my experience, if you have non-specific staining of your antibody, it will not go away if you increase the sensitivity of your detection method. It might even make the background problem worse.
Do you know if your Ab works on fixed tissue samples? If so, I would suggest playing around with Ab concentrations and using isotypic Abs as blockers.
Hi
Yes I know people have used this antibody on mouse and human fixed tissue but their protocol and their dilutions vary a lot (10 fold). They use a 4% PFA fixation for 10min.
I will try to use isotypic Abs to block.
Thanks
Dear Florian,
The avidin/biotin method is quite good and probably the most specific IHC protocoll. On the other hand overnight incubation (4°C) (if you have not already used it that way) of the primary antibody can give you much better results. Excessive rinsing with PBS of the primary ab can also decrease background. If you use DAB/H2O2 at the end of the protocoll the usage of TrisHCl instead of PBS as buffer for washing steps can also improve the outcome. Sometime simply the secondary antibody is responsible for aspecific signals. For example if you use it in a species related to the secondary antibody's host species (e.g using rat secondary ab on mouse tissues). Of course the latter won't apply If you work on cell cultures instead of tissue sections. You can apply as well blocking agents like serum 1% BSA or 2% goat serum before and after the primary, but there is still an ongoing scientific debate whether these serum blocking is effective or not.
If avidin/biotin method still does not work try the peroxidase-anti-peroxidase method (PAP) that is highly specific and sensitive (and also works for semithin sections), but can be used just with rabbit primaries.
Hope I could help.
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