Re-staining Immunohistochemical slides
Posted 22 June 2011 - 08:49 PM
I was just wondering if anyone has done a re-stain immunohistochemical tissue slides before. If so, how can it be done? Is there a protocol that I can refer to?
Posted 22 June 2011 - 09:00 PM
Posted 24 June 2011 - 12:18 AM
Posted 26 June 2011 - 02:43 PM
What do you mean with "wrong"? Like, your primary was raised in rabbit, but you put an anti-Rat secondary? Then you can just do the same procedure again, but with the right secondary. Or do you want to know if you can strip the signal and re-stain? Getting rid of your signal and do a new stain? There are several methods, like stripping, photobleaching and so on...
I stained the anti-rabbit primary with an anti-mouse the first time. Washed it with TBST overnight and restained it with a mixture of Goat anti-rabbit and Donkey anti-goat mixture of secondary the second time. Will this still work? Can I do another staining again still?
PS: The secondary antibody mixture was made by mixing the 2 antibodies together in the same tube before applying onto the tissue samples.
Posted 26 June 2011 - 11:37 PM
Posted 26 June 2011 - 11:49 PM
You don't need to wash the anti-mouse Ab off, it should not stain the sample (only if you're working with mouse tissue, are you?). Usually you can use only anti-rabbit secondary, using also anti-goat Ab will only increase your background. But the staining should still work.
Thank you for the reply Rsm. I'm working on human tissues so it's not going to be a problem. But yes, I did get quite a bit of background but the staining worked and the peptide blocking as well.
I should clarify that I do mix my secondary antibodies together in some experiments (usually when I have two different primary antibody in one section) to get different colors in my result. The mistake here is that I mixed an anti-rabbit raised in Goat and an anti-Goat raised in Donkey secondary antibody together in one tube thus the background.