I am doing ICC with chicken brian sections. I used slide mounted sections and I did antigen retrieval with tris buffer ph 9.5. Then i used hydrogen peroxidase to quench endogenous activity, blocked with 5% normal goat serum, incubated with primary ab raised against rabbit for overnight, secondayr antibdoy was biotinylated igG (goat anti rabbit) and finally incubation with ABC-Complex. I did glucose oxidase Ni DAB staining. Usually when I do the staining I get staining in less than 2 minutes. This time i didnt get any staining, inspite of longer incubation times. I thought it could be because of antibodies, I repeated the expt with a fresh batch of antibodies. Still I didnt get any staining, In fact there is no background in sections. Do you any of you have an idea why is it so???
I also tried to add stain to substrate and it did change in color.
