Dear Sanjaykumar,

Not necessarily.  I assume that you are using DAB as a chromogen to detect an HRP-linked secondary antibody or ABC reagent?  If so, you may not have sufficiently inactivated the endogenous peroxidase in the tissue.  This can happen if the H2O2 is old.

If you post your IHC protocol, it would be easier for us to help you troubleshoot your problem.

Cheers!

Jill

Thanks Jill....

I will send you.

Dear Jill, Good morning.

My IHC protocol is as follows.

De-parafinization in xyline; Re-hydration in different percentage of alocohol; Antigen retrival in water bath for 95 degree for 30 min; peroxidase block for 20 min; protein block for 5 min; Primary antibody (polyclonal) for 30 min; secondary antibody for 30 min; DAB for 4 min; counter stain haemotoxylin for 20 min; again de-hydration in different grades of alcohol; clearing in xyline for 5 min. All the steps are carried at RT.

waiting for your reply.

Sanjaykumar... 

Dear Sanjaykumar,

For the first step in trouble shooting your background staining, we need to know if you have a light brown background on your negative control slides where you have omitted BOTH the primary and secondary antibody.  If you do, you may still have active peroxidase in the sections.  I always make sure that I see little bubbles on the tissue when I perform the peroxidase blocking step.  If you do not, you should replace the H2O2.  If you do, you may just need to increase either the time of the block or the concentration of H2O2.  It is easier to tell if you have background staining during troubleshooting if you omit the haemotoxylin staining before dehydration and coverslipping.

If the sections that lack both the primary and secondary antibody are free of non-specific background staining, then the next control to check is the background when you omit the primary antibody from the procedure.  If you see background with this type of negative control, it suggests that you secondary antibody is binding to some endogenous epitope in your tissue.  You may need to either 1) increase your concentration or time with the protein block, 2) change the type of protein used in your blocking solution, or 3) reduce the concentration of your secondary antibody.  Personally, I block for at least 30 minutes at RT, but I work on 10 um cryostat sections.  Remember to drain off the blocking solution, rather than rinsing the sections.

If the sections with only secondary antibody lack non-specific staining, then it suggests that your primary antibody is binding non-specifically.  This can be from using too concentrated a primary antibody solution during incubation, or from insufficient blocking or rinsing.  I personally prefer to titer my primary antibodies for incubation at 40 C overnight.  My secondary antibodies are titered for binding for 1 or 2 hours at RT, and I have a control primary antibody that I use for determining this with new lots or suppliers of secondary antibodies.  I work on adult brain, so I use rabbit anti-GFAP from DAKO as my control primary antibody for anti-rabbit secondary antibodies.

I do not use antigen retrieval,  so I don't know if there are any concerns with that step.

I have also played with different ionic strength buffers to reduce background: https://www.rndsystems.com/resources/protocols/preventing-non-specific-staining

If none of the above changes help, please let me know and I will send the buffer recipes.

Good Luck!,

Jill

Dear Jill, Thanks for your valuable suggestions. I do have increased the peroxidase step and also the protein block step. Now I am getting better results.

Thanks once again for your time & be in touch

Sanjaykumar...