No staining in immunohistochemistry

Explore no causes for no staining in immunohistochemistry.

Why is there no staining?

Possible causes
Solution
Antibodies or amplification kits may have lost activity due to improper storage and handling

  • Check the storage instructions for your products on the datasheet.

  • Avoid excessive freezing / thawing.

  • Run a positive control.

If target protein is a membrane protein, permeabilization has damaged cell membranes.

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

  • Remove the permeabilizing agent from your buffers.

If target protein is a nuclear protein, the antibody cannot penetrate the nucleus
  • Add a strong permeabilizing agent like Triton X to the blocking buffer and antibody dilution buffer.
If tissue is embedded in paraffin, deparaffinization may be insufficient
  • 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.
If using formalin and paraformaldehyde fixatives, fixation procedures may be masking the epitope

  • Use different antigen retrieval methods to unmask the epitope (eg heat mediated with pH 6 or pH 9 buffer, enzymatic, etc).

  • Fix sections for less time.

Not enough antibody is bound to the protein

  • 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 type of IHC you are using (eg formalin/PFA fixation, fresh frozen, etc).

  • Test the antibody in a native (non-denatured) western blot to make sure it is not damaged.

The primary antibody and the secondary antibody are not compatible

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

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

The protein of interest isn't present in the tissue

  • Check the scientific literature to see if protein is expected in your tissue type.

  • Run a positive control.

The protein of interest is present in low abundance
  • Use signal amplification to maximize the signal eg a biotin-conjugated secondary antibody.
The buffer is contaminated with bacteria

  • Add 0.01% azide to the antibody storage buffer.

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

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