Hello to all Researchers,
I would like to ask for your advice. I am performing an IHC analysis using a primary antibody (host: rabbit) and a secondary antibody (goat anti-rabbit). I use perhydrol solution in methanol or blocking solution (ready to use). I block with goat serum 5%. I stain using DAB and contrast with haematoxylin. In the negative control (omitting the first-strand antibody), DAB stains the whole slide uniformly (photo attached). I have tried changing incubation times and concentrations, but to no avail. Has anyone encountered something like this and would like to share a solution to this problem?
All the best.
Hello,
The uniform faint staining is negligible but that depends on how your actual staining with the specific antibody looks. Can you share a picture of your positive staining for comparison? Also, does your wash solution contain a detergent?
While taking the images, decrease the exposure time and laser intensity.
If you are still facing the same problem, adjust the background using ImageJ, you are gonna find so helpful tools.
Another suggestion, (I read but have not used) is to take another image for the same area without laser light, then by ImageJ you subtract the phase image from the DAPI one to get rid out of the background.
Did you do the H2O2 endogenous peroxide blocking step? If not, you should try it!
Yi-Chun Cheng Of course, that's what I did at the start.
As I have written all the steps of blocking I have done.
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Thank you all for your advice. I think I have partly got rid of the problem by incubating in H202 for longer. In addition, I increased the amount of xylenes and alcohols. All the best.
Hey there, I'm facing the same problem right now. Have you figured out the problem?
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