Complete absence of IHC signal due to reagent degradation (Immunohistochemistry (No Staining))

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Accepted Answer

No staining is observed in immunohistochemistry despite proper protocol execution. Previously validated antibodies or detection kits fail to produce any signal. Common causes: 1) Antibodies or amplification kits lost activity due to improper storage conditions 2) Excessive freeze-thaw cycles degraded antibody or reagent functionality 3) Fluorophore-conjugated secondary antibodies damaged by light exposure (photobleaching) 4) Buffer contamination with bacteria compromising reagent integrity Solutions: 1) Verify storage instructions on product datasheet and maintain proper storage conditions 2) Avoid excessive freeze-thaw cycles; aliquot antibodies for single use 3) Run positive control with previously validated primary antibody 4) Test primary antibody in native western blot to confirm it is not damaged 5) Keep fluorophore-conjugated secondary antibodies in the dark at all times 6) Add 0.01% azide to antibody storage buffer and use fresh sterile buffer (e.g. sterile PBS)