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.

Hydrophobic interactions of the antibody and proteins in the tissue
Lower the salt concentration of the antibody stain buffer.
Tissue sections have dried out
Examine the tissue; sections with higher background staining at the edges than towards the center are often dried out. Prevent tissue sections drying out by keeping them in a humidified chamber.
Non-specific binding of primary antibody
Use the blocking step just prior to primary antibody incubation.
Non-specific binding of secondary antibody
Use an antibody that has cross-reactive IgG species removed (pre absorbed against sample species).
Ionic interactions
Ensure to change buffers.
Antibody is binding to Fc receptors on the target's cell surface
To block open binding sites, use 10% serum of the host secondary antibody.
Incubation temperature may be too high
Be sure to incubate at a temperature of 4°C.
Tissue inadequately washed
Include washing steps with fresh PBS/TBS.
Endogenous peroxidase
Use a peroxidase quenching buffer in blocking step before primary antibody.
Permeablization of membrane
Be sure to remove detergents from buffers.
Fixative was too strong
The epitope was modified, so be sure to change the antigen retrieval method.

Antibody incubation too long
Lessen the primary antibody incubation time.
Endogenous peroxidase
Use a peroxidase quenching buffer in the blocking step before the primary antibody.
Sections have dried-out
Ensure to keep slides in a humidity chamber.
Delay in fixation
Be sure to fix the tissue immediately.
Insufficient washing/blocking
Increase the washing time and number of washes.

Primary antibodies too concentrated
Dilute primary antibody solution. Perform a titration to determine optimal antibody dilution.

Hydrophobic interactions of the antibody and proteins in the tissue
Tissues have dried out
Cover the tissues in liquid at all times during the experiment.