Dear Manjit,

It seems your blocking step is somehow not sufficient to counteract the non-specific interaction of your primary antibody. Check in the following thread of RG for some related answers.

https://www.researchgate.net/post/In_immunohistochemistry_which_serum_do_you_mix_to_the_secondary_antibody_to_reduce_aspecific_staining

Cheers,

Hi,

1- check the PH of sodium citrate

2- use detergent based on your marker (tween or triton)

3- increase the duration of blocking

4- decrease the DAB exposure to the lowest

5- in my personal experience when I had too much background I put unstained slides for an hour in sun light and I got much better staining results.

let me know if you could not get good qulity staining I will share my protocol with you.

Best,

Masoumeh

Thank you @Sebastian and Masoumeh for your input.

Masoumeh Majidi Zolbin

-The pH of the citrate buffer is 6.0. ButI'll check again before doing antigen retrieval step.

-I have been using the detergent Tween 0.05% in TBS for washing and as a diluent for the primary antibody. Triton might be little harsher.

-should I go 2 hr of blocking with goat serum? or increase the percent of goat serum from 10-15%

- i have tried the various exposure of DAB. it did not help.

-putting the unstained slides in sunlight for 1 hrs. Is that even before doing the xylene steps?

Thanks again.

If your negativ controls showed no reaction, then the reason for unspecific background staining seems to be the primary antibody. In that case I would recommand to run your immuno protocol without antigene retrievel. Use different concentrations of the primary ab to find out which is the best. To intensify the staining result, if it is nessary, you can use instead of DAB DAB-Ni. Ni-ammoniumsulfat enhances the staining.

Thank you Ute Neubacher for the suggestion.

To me also, the unspecific background seems to be coming from primary. But why IgG also turns brown after DAB.

What is the principle of doing IHC without antigen retrieval and then using DAB-Ni rather than DAB only?

Thanks again.

Generally unspecific background staining could have different reasons. To find out wheather your primary ab or your secondary or detctions system is responsible for this fenomena you have to check this with negativ controls. I recommand to use negative controls without primary ab and without replacement by IgGs (in my experience IgGs can also produce unspecific background staining). Sometimes polyclonal abs show unspecific bindings while the negative control is negativ. Look into the datasheet. If this ab was tested by western blotting and the blot shows different bands additionaly to those specific band then this other bands could be responsible for unspecific bindings in your tissues.

The principle of antigene retrieval is heat induced antigene demasking. That means: antigene retrieval in citrate buffer (pH 6) or EDTA-buffer (pH8)

removes the strong crossling of proteins by aldehyde fixation, so that the antibodies can discover the epitops in a better way. But somtimes this enhancement techique is to much. That's why I recommand to do your immuno without antigene retrieval. May be you have to use higher primary ab concentrations --> titrations of optimal concentration, because only the epitopes which are in the surface of the tissue sections will be bind by the ab. To increase the contrast you can use DAB-Ni (blue-black color) instead of DAB (brown). Good luck!