Complete Absence of IHC Staining (Immunohistochemistry (CST Guide))

Question

Accepted Answer

Complete lack of staining observed in immunohistochemistry experiment despite using validated antibody. Tissue sections appear negative even when positive control should show signal. Common causes: 1) Antibody not validated for IHC application or tissue type 2) Sample is truly negative for the target protein (especially for phospho-specific antibodies or rarely expressed proteins) 3) Slides stored improperly or for extended periods; protein epitopes degraded during storage 4) Tissue sections dried out during staining procedure; sections not kept covered in liquid throughout protocol 5) Inadequate deparaffinization causing spotty, uneven staining that appears as no signal 6) Chemical crosslinks from fixation preventing antibody access to masked antigen targets Solutions: 1) Verify antibody is validated for IHC and use high-expressing positive control (paraffin-embedded cell pellets) 2) Confirm sample expression status; accept that phospho-antibodies may not stain 100% of cases 3) Use freshly cut slides before experiments; if storage necessary, keep at 4°C without baking 4) Ensure tissue sections remain covered in liquid throughout entire staining procedure 5) Repeat experiment with new tissue sections using fresh xylene for deparaffinization 6) Optimize antigen retrieval: use microwave (preferred) or pressure cooker, not water bath; prepare fresh 1X buffer solutions daily