Hello alI.
I ´m doing IHC on brain slices fixed on paraffin, and I have a question regarding with DAB reaction. so.. When I prepare DAB reagent on water ( DAB 1%+ 0.03% H2O2 all in mq water) WITHOUT quench endogenous peroxidase, I have a good stain of my target, and a lower background. As negative control I'm using slices without primary antibody, and I don´t see any signal there. so, apparently I have a specifical signal. But, when I prepare DAB reagent on PBS 0.01M instead to water, the specific staining dessapear, and come back the background.... so, my question is, why this happen??. Can I belive in my result?
Dear Valeria,
As I remember, both water and PBS can be used to prepare solutions and work. But do you use just Water or just PBS to prepare ALL solutions, including the antibody dilution (or if it is RTU, in which solution it is solved).
May be it is about the pH, as some of the treatment solution are pH~6.9, while other pH~8-9? all the steps of tissue treatment and staining should be done in similar conditions, so similar pH should be respected.
I have some experience with Dako products and, generally, this was one of the main points.
At the same time, in some cases water quality is an issue, as non processed water would stimulate specific staining, but I am not aware if it works in all tissues.
So check if all the steps from tissue processing until staining were performed in similar conditions.
Good luck!
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