Hi!
My question is how to troubleshoot the triple immunohistochemical staining.
I have two well-validated antibodies, stained with Alexa Fluor 488 and 546, respectively.
Together, they work very good. But today I decided to add a third antibody, in 647 (in a different species). When stained all three together, the '546' antibody stopped working (extremely faded and unspecifc). And in the same batch, double-staining controls 488/546 worked amazingly well.
All three antibodies are in different species: mouse, rabbit, and guinea pig.
Please, advise, I'm new in this field :/
Thank you!
You need to control the "species" in all antibodies, primary AND secondary, and control for overlaps, as well as what the primary antibodies can detect (e.g. detects human, mouse or only human etc.). This information is needed to understand what is happening in this case.
I agree with Elizabeth, do it sequentially. Rabbit-guinea pig may be cross-reacting or somehow competing for the secondary. You could even re-fix the cells (5 min) after the first doublet of primary/secondary antibody pair , block again 15 min and proceed with the third primary/secondary pair.
Thank you a lot for your comments!
I will try sequential staining today.
Related thing: is there any point in using “IgG1 cross-adsorbed secondary antibody” instead of regular “IgG secondary antibody” for monoclonal primary IgG1 in brain? I assume there is no IgGs in brain due to BBB?
As Elizabeth E LeClair
said, do it sequencial buth in both ways, where the 546 comes first (together with 488), and another slide second (after 647). If the primary bind the same cell compartment/structure or even sequence on the protein, it might compete.- Badges
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