We have been trying to optimize IHC. We are using citrate buffer ph:6 for antigen retrieval in microwave oven for 20-30 minutes. Giving 1 hour incubation for primary antibody and 40 minutes for secondary antibody. But we are unable to get results. Hematoxylin staining is working fine but DAB is not going inside cells giving these brown spots. Attached are the images of recent try.
Thanks in advance.
hello Rida Mudassar
first I recommend you to contemplate the following questions:
What is the primary antibody you are using and why?
What is your detection system?
Are you using the technique controls specific for your marker?
In which tissue find basal expression and in which not?
if all these issues are already contemplated, it would be convenient to review the quality of your reagents or compare your protocol with other protocols that use the same marker because this marker looks unspecific or I don´t appreciate it well maybe of the quality of the image.
I hope it is of some help to you
Hi Rida Mudassar, are you using anything to permeabilize the cells? I typically prepare my primary antibody within PBS + 0.1% tween or PBS + 0.2% Triton-X. You could also try extending your primary antibody incubation time, as well as the washes, secondary, and DAB staining time.
You should also consider having a positive control to assess whether the methods are being performed correctly. Hope this helps.
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