Lack of antigen
Check protein expression by in situ hybridization.
Improper storage of antibodies
Follow storage instructions on the datasheet. Aliquot antibodies in a sufficent volume 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
Check antibodies independently on a dot blot.
Inadequate tissue fixation
Increase or decrease fixation incubation, and try to change the fixative.
Over fixation
Reduce the duration of the immersion or post-fixation steps and be sure to include the Antigen Retrival step.
Antigen destroyed prior to staining
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 embedding or fixation
Embed tissue at 58 °C or below. Try restoring immunoreactivity through various antigen retrieval techniques.
Incomplete deparaffization
Use fresh xylene and increase xylene incubation time.
Insufficient antibody incubation
Incubate primary antibody overnight at 4°C
Photobleaching
Ensure to proceed with all steps in the dark.
Antigen retrieval was ineffective
Try to change the incubation time and also change solutions.
Antibodies are not compatible
Make sure you use a secondary antibody that was raised against the primary antibody species. Make sure that the isotypes of the primary and secondary are compatible.

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