||Test or Action
|Lack of antigen.
||Check protein expression by in situ hybridization (in some rare cases translation may be blocked even though mRNA is detected).
|Antibodies do not work due to improper storage.
||Follow storage instructions on the datasheet. In general, aliquot antibodies into smaller volumes sufficient to make a working solution for a single experiment. Store aliquots in a manual defrost freezer (-20 to -70 °C) and avoid repeated freeze-thaw cycles.
|Inactive primary or secondary antibodies.
||Test reporter system independently to assess reagent viability.
|Inadequate tissue fixation.
||Try increasing the fixation time or try a different fixative.
||Reduce the duration of the immersion or post-fixation steps. If immersion fixation cannot be avoided (for example, collection of postmortem tissues or biopsies in pathology lab), antigens may be unmasked by treatment with antigen retrieval reagents.
|Incompatible secondary and primary antibodies.
||Use a secondary antibody that will interact with the primary antibody. For example, if the primary antibody was raised in rabbits, use an anti-rabbit secondary antibody.
|Antigen was destroyed before incubation with the primary antibody.
||If quenching of endogenous peroxidase was done prior to the addition of primary antibodies, block peroxidase after incubation with the primary antibody.
|Epitope altered during fixation or embedding procedure.
||Try restoring immunoreactivity through various antigen retrieval techniques.
Embed tissue at 58 °C or below.
|Antigen retrieval was ineffective.
||Increase the time of treatment or change the treatment solution.
|Reagents omitted or used in wrong order.
||Repeat staining and confirm that correct reagents are used and are added in the correct order.