Hi, I recently started doing a lot of IHC and I’ve been staining mouse coronal section (not in parafin) with 3D6 antibody to visualize plaques. My problem is that the background is too dark and plaques are not dark enough to have enough contras. I develop with Dab for only 35sec, and use ABC elite kit from Vector lab prior. Anyone has any suggestions?
Dear Dominika Pilat ,
I sounds a little bit as if you have still some background activity from internal peroxidase. Have you tried to stain an unstained control with DAB? If you have a brown staining there you should take action to diminish the peroxidase activity in the slice. Usually with H2O2 treatment before the Antibody staining. If you would still have some background you might think about unspecific secondary antibody binding (which is also easy to test). If that is the case try to lower the sec. antibody.
When you would have optimized your background, you can hopefully also increase the incubation with the DAB and thereby getting a stronger signal for your antibody staining.
Best wishes
Soenke
- Badges
- Science method