I am combining 2 antibodies using IHC multiplex double stain, any one has a suggestion. How can we increase the signal of the staining. I have tried to increase the concentration of the antibodies and the chromogen, however there are no effect.
I don't know wether you makes whole mount immunostaining or slices.
For whole mount immunostaining, After incubation of your first antibody, you can try to wash them using PBS for a long time. For example, washing them 1h twice.
M Musleh Althobiti you're developing the signal with a chromagen, correct? Not florescent? Does the signal look better when labeling only one at a time?
My overall recommendation would be to invest in Vector ABC kits (avidin-biotin-complexing) if you're having issues with signal. I use them routinely and have beautiful results with very low background. You also end up using less primary antibody, so you can offset the kit costs depending on how often you're performing IHC. However I would carefully do antibody titration experiments (inc negative controls) with each antibody one at a time before you try co-labeling. I would do this no matter what method, but especially with ABC because it can amplify your signal quite a bit, and you want to make sure you're labeling the correct cell compartments, etc.
It is more tricky to co-label with ABC, but it is possible.You have to do sequential labeling with some extra blocking steps, but it can be done. Vector has a pretty good guide which I attached here. Not sure what species you're in, but regardless I also suggest pre-adsorbed secondary antibodies, especially when co-labeling! And I do not work with Vector in any way, but have used their products for years.