DAB staining sometime did not work for me because I think problem of H2O. If H2O used for DAB making solution was old or kept in bottle for long (what happened in my case), the DAB staining might not work.
I think you use you slides again with fresh DAB. I usually use whole mount embryonic tissue sample for DAB staining. And after 2-antibody can keep then O/N. The next day DAB staining works well for me. Thanks.
Thanks for your replies.
@ Md Riaj Mahamud : I did use fresh de-inoized water, so I don't think that was the issue in my case.
@ Subuhi Abidi: I did make up the ABC mixture 30 minutes before use, and prepared the DAB solution just before use.
No reactivity with DAB but staining with another type of substrate can be due to the sensitivity of the chromagen or peroxide as was mentioned. One really should use a range of primary antibody concentrations when changing a detection system. Each one has a very different range of efficacy. I recently tested Vector Red too but got NOTHING with it compared to the DAB substrate, I would bet my bottom dollar on the fact that the VR was not sensitive compared to NiDAB. This article can give you an idea of how different the detection can be on the same tissue with the same primary etc.
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