No staining in immunohistochemistry

• Check the storage instructions for your products on the datasheet.
• Avoid excessive freeze/thawing cycles.
• Run a positive control with a primary antibody previously validated with your protocol.
• Test the primary antibody in a native (non-denatured) western blot to make sure it is not damaged.

• Use a less stringent detergent (eg Tween 20 instead of Triton X).

• Remove the permeabilizing agent from your buffers.

• Add a strong permeabilizing agent like Triton X to the blocking buffer and antibody dilution buffer.

• Deparaffinize sections longer and use fresh xylene.

If using fluorescent detection, the fluorophore may have been damaged.

Exposure to too much light can lead to photobleaching.

• Always keep fluorophore-conjugated secondary antibodies in the dark.

• Use different antigen retrieval methods to unmask the epitope (eg, heat-mediated with pH 6 or pH 9 buffer, enzymatic, etc).
• Optimize the antigen-retrieval incubation time.
• Fix sections for less time.

• Add a higher concentration of the primary antibody.

• Incubate the sample for longer with the antibody (eg overnight) at 4°C.

The antibody may not be suitable for IHC

IHC procedures reveal the protein in its native state, which not all antibodies are suited to.

• Check the antibody datasheet to see if the antibody has been validated in the IHC application and tissue preparation you are using (eg, FFPE, fresh frozen, etc).

• Ensure the isotypes of the primary and secondary are compatible.

• Use a secondary antibody that was raised against the primary antibody species.

• Check the scientific literature to see if protein is expected in your tissue type.
• Use a known positive tissue control.

• Use signal amplification to maximize the signal eg a biotin-conjugated secondary antibody.

• Add 0.01% azide to the antibody storage buffer.

• Use fresh, sterile buffer (eg our sterile PBS).