Strong positive background / non-specific staining (Immunohistochemistry (IHC))

Question

Accepted Answer

Whole slide appears brown/diffuse stained; negative control also shows signal; impossible to distinguish true positive signal. Common causes: 1) Primary antibody concentration too high 2) Antigen retrieval was too aggressive or wrong protocol 3) Inadequate blocking 4) High residual HRP activity in detection system 5) DAB color development time too long Solutions: 1) Reduce primary antibody concentration or shorten incubation time 2) Optimize antigen retrieval (time, temperature, buffer) 3) Strengthen blocking (use normal serum + BSA appropriate for secondary antibody host) 4) Use high-quality secondary antibody and detection system 5) Shorten DAB development time; monitor under microscope