Re-staining Immunohistochemical slides - (Jun/22/2011 )
Hi,
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?
I forgot to mention that these tissue samples are precious as we don't have many of them left. I've put on the wrong secondary antibody twice and is wondering if there's anyway that I can salvage them.
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...
Rsm on Fri Jun 24 08:18:09 2011 said:
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.
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.
Rsm on Mon Jun 27 07:37:31 2011 said:
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.