Inadequate Washing Steps Causing Poor Contrast (Immunohistochemistry (CST Guide))

Question

Accepted Answer

High background persists with poor contrast between positive signal and background despite other optimization steps, making interpretation difficult. Common causes: 1) Washing slides fewer than 3 times after antibody incubations 2) Washing duration less than 5 minutes per wash step 3) Using incorrect wash buffer (not 1X TBST #9997) 4) Insufficient agitation or mixing during wash steps Solutions: 1) Wash slides exactly 3 times for 5 minutes each with 1X TBST (#9997) after primary antibody incubation 2) Wash slides exactly 3 times for 5 minutes each with 1X TBST (#9997) after secondary antibody incubation 3) Use only recommended 1X TBST (#9997) as wash buffer; prepare fresh if needed 4) Ensure adequate agitation during washing to remove unbound antibodies effectively