It looks like there is lot of non-specific binding. 5% BSA should be enough concentration to block.

1) Have you titrated your antibody? may be concentration is too high and making it non specific.

2) Do you have isotype control to try non-specific binding?

3) how are you diluting your antibody? i recommend dilute in BSA-PBS.

Suggestions:

-try using 0.1% tween in PBS for washing steps. and BSA in PBS-tween for blocking.

-I think it is something related to your fixation step. acetone damages membrane hence the membrane protein expression might not be well.. try using 4% PFA for fixation..

Finally, PFA-> use PBS-Tween to wash -> dilute antibody in 1% BSA-PBS-tween to avoid non-specific binding.

Good luck though..

Thanks Matti Ullah I'll have to try PFA instead of acetone and adding tween for my washes. I'm using antibody concentrations from literature between 5 ug/mL for one and 25 ug/mL for the other, but I could always cut it down. I'm currently just diluting in PBS so doing the BSA-PBS-tween mixture could help too.

The same answer of Corrine Nief

Does your tumor section have high WBC infiltrates? Lymphocytes, monocytes, and macrophages themselves may catch random antibodies through their Fc Receptors which can cause false positive staining. Hence, I suggest you do FCR blocking first when staining WBC’s.

You may want to perform antigen retrieval step if your specimen is FFPE.

Creo que es todo un acierto la indicación de Matti Ullah con respecto a la fijación con PFA.