High Background from Excessive Antibody Concentrations (Immunohistochemistry (High Background))

Question

Accepted Answer

Elevated background staining throughout tissue sections due to excessively high concentrations of primary or secondary antibodies leading to non-specific binding. Common causes: 1) Primary antibody concentration exceeds optimal working dilution 2) Secondary antibody concentration too high for the application 3) Lack of antibody titration to determine optimal concentration 4) Non-specific binding of secondary antibody to tissue components Solutions: 1) Dilute primary antibody further to its optimal concentration through titration 2) Dilute secondary antibody to its optimal concentration 3) Run a control without primary antibody to assess secondary antibody background 4) Use pre-adsorbed secondary antibody against the immunoglobulin of sample species 5) Ensure secondary antibody is raised in a different species from the sample