Immunohistochemistry Troubleshooting

Immunohistochemistry is a complex experiment with multiple parameters that need to be considered and optimized. The following troubleshooting guides are intended to explain the causes and possible solutions for common problems in immunohistochemistry/immunofluorescence (IHC/IF) staining.

No Staining

Possible Problems	Recommended Solution(s)
Inadequate or excessive 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 (e.g. collection of postmortem tissues or biopsies in pathology lab), antigens may be unmasked by treatment with antigen retrieval reagents.
Epitope altered during fixation or embedding procedure	Try different antigen retrieval methods to restore immunoreactivity. Embed tissue at 58°C or below.
Insufficient deparaffinization	Extend the deparaffinization time and replace with fresh xylene.
Lack of antigen or insufficient	Run a positive control or use an amplification step to maximize the signal.
Antigen retrieval was ineffective	Increase the time of treatment or change the treatment solution.
Not enough primary antibody	Increase the concentration of primary or secondary antibodies. Incubate for longer periods of time (e.g. overnight) at 4°C.
Incompatible secondary and primary antibodies	Use a secondary antibody that was raised against the species in which the primary was raised (e.g. primary is raised in rabbit, use anti-rabbit secondary).
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 freezer (-20 to -70°C) and avoid repeated freeze-thaw cycles.

Inappropriate Staining

Possible Problems	Recommended Solution(s)
Fixation method is inappropriate for the antigen	Try a different fixative or increase the fixation time.
Antigen retrieval may be inappropriate for this antigen or tissue	Try different antigen retrieval conditions.
Electrostatic charge of the antigen has been altered	Try adjusting the pH or cation concentration of the antibody diluent.
Delay in fixation caused diffusion of the antigen	Fix tissue promptly. Try a cross-linking fixative rather than organic (alcohol) fixative.

High Background

Possible Problems	Recommended Solution(s)
High concentration of primary and/or secondary antibodies	Titer antibody to determine optimal concentration needed to promote a specific reaction of the primary and the secondary antibodies.
Non-specific binding of primary and/or secondary reagents to tissues	Use blocking step just prior to primary antibody incubation (we recommend blocking with 10% normal serum for 1 h for sections or with 1-5% BSA for 30 min for cells).
Non-specific binding of secondary antibody	Run a secondary control without primary antibody.
Tissue dried out	Avoid letting the tissue dry during the staining procedure.
Active endogenous peroxidases	Use enzyme inhibitors (e.g. Levamisol (2 mM) for alkaline phosphatase or H₂O₂ (0.3-3% v/v) for peroxidase).