Weak staining / signal too faint (Immunohistochemistry (IHC))

Question

Accepted Answer

Positive regions show very pale staining; barely distinguishable from negative control; signal appears dotted or discontinuous. Common causes: 1) Primary antibody concentration too low 2) Antigen retrieval was insufficient 3) Primary antibody incubation time too short 4) Antibody is degraded or stored incorrectly 5) DAB development time too short Solutions: 1) Increase primary antibody concentration or extend incubation time 2) Optimize antigen retrieval (try longer time or different buffer) 3) Extend primary antibody incubation (4 °C overnight gives best results) 4) Check antibody validity and storage conditions; aliquot to avoid freeze-thaw 5) Extend DAB development time while monitoring under microscope