Hi ALL,
I am trying to stain my paraffin embedded brain tissue sections with DAB stain and a counterstain. My DAB works however my counterstain doesn't work. These are my images attached below. I cannot detect color in these images. Here is my protocol:
Deparaffinize and dewax
Antigen retrieval and Hydrogen peroxide incubationss.
Primary Ab overnight
Next day secondary antibody and then DAB staining with the Vectastain kit.
I used Hematoxylin Blue here as a counterstain and imaged with brightfield.
The images you posted appear to be grayscale. Do you see brown DAB staining using the the microscope?
How does a negative reagens control look like? Are there any blue nuclei?
If DAB-staining is very intense, it may mask the binding sites of hematoxylin.
Have you got the right hematoxylin? correct incubation time?
Hi, I agree with Gundrun. You should just try staining one of your sections that aren't IHC stained to see if hematoxylin even works. As far as I can see your IHC stains something nuclear in neurons. Even tho you woudn't expect many glia nuclei, there should be at least a few that are stained blue with hematoxylin if it is working.
To cut the long story short: Try staining a sample without IHC, see if nuclei are blue. If they are, IHC is covering most of the counterstain (you could incubate for longer in hematoxylin or develop DAB for shorter). If they aren't blue, your hematoxylin is probably old and oxidized.
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