I am doing RBC staining with Glycophorin A. It gives very nice and crispy brown stain when I check it after DAB+Chromogen reaction but it turns weirdly greenish after the hematoxylin-eosin stain. Please suggest if you have any idea.
Those RBCs don't appear to have been stained at all, or rather that is how RBCs appear on a slide stained only with hematoxylin. Are you using DAB as your chromogen or some other HRP substrate (such as AEC)? DAB shouldn't wash out during counterstaining, washing, and dehydrations, but other chromogens can.
Hello, good afternoon, I hope you have already solved your problem; However, what you observe usually happens when you put eosin, remember that IHC only contrasts with hematoxylin, preferably Gill or Mayer.
John Hardy Lockhart Hi, the RBCs appeared well stained after I used checked for the DAB choromogen reaction. But later after I did the H&E staining it turned green
Can you give more details on the steps you perform after the DAB development?
John Hardy Lockhart
Sure,
After confirming DAB colour development by looking in the microscope, I placed the slides in TBST to stop the reaction, then in 70% alcohol (3 mins), 70% alcohol (3 mins), 96% alcohol (3 mins). Followed by putting them in xylene three times (5 minute each) Then I mounted the slides with cover slips.
Francisco Javier García Vázquez The problem still remains :D
I am using Mayer's Hematoxylin by Bio-optics.
You are inhibiting endogenous peroxidase with 0.9% H2O2 in water or methanol?
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