Problem: Lack of Staining
Possible Source
Lack of antigen
Test or Action
Check protein expression by in situ hybridization (in some rare cases translation may be blocked even though mRNA is detected)
Possible Source
Antibodies do not work due to improper storage
Test or Action
Aliquot antibodies into smaller volumes sufficient to make a working solution for a single experiment. Store aliquots in a freezer (-20 to -70°C) and avoid repeated freeze-thaw cycles
Possible Source
Inactive primary antibodies
Test or Action
Replace with a new batch of antibodies
Possible Source
Inadequate tissue fixation
Test or Action
Try different fixative
Possible Source
Tissue overfixation
Test or Action
Reduce duration of postfixation or fix tissues at 4° C. If postfixation cannot be avoided (for example, collection of postmortem tissues in pathology lab) antigens may be unmasked by treatment with antigen-retrieval reagents
Possible Source
Incompatible secondary and primary antibodies
Test or Action
Use secondary antibody that will interact with the primary antibody. For example, if primary antibodies were raised in rabbits, use anti-rabbit secondary but not anti-mouse or anti-goat
Possible Source
Inactive secondary reagents
Test or Action
Replace with new reagents
Possible Source
Antigen was destroyed before incubation with primary antibody
Test or Action
Quenching of endogenous peroxidase was done prior to the addition of primary antibodies. Block peroxidase after incubation with primary antibody