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Staining in paraffin-embedded mouse cerebellum tissues by immunohistochemistry

Troubleshooting and using controls in IHC

Detailed troubleshooting tips and techniques for IHC.

Common problems

Common problems

​No staining

Browse the table below for possible causes of no staining, and how to fix this — or prevent it altogether.

Possible causes

Solutions

The primary antibody and the secondary antibody are not compatible

• Make sure you use a secondary antibody that was raised against the primary antibody species (eg primary is raised in rabbit, use anti-rabbit secondary).
• Make sure that the isotypes of the primary and secondary are 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.
Not enough antibody is bound to the protein
  • Add a higher concentration of primary antibody
  • Incubate the sample for longer with the antibody (eg overnight) at 4°C.
The antibody may not be suitable for IHC procedures which reveal the protein in its native state
  • 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.
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.
The protein of interest isn't present in the tissue
  • Run a positive control.
  • Check the scientific literature to see if protein is expected in the tissue type.
The protein of interest is present in low abundance
  • Use signal amplification to maximize the signal, eg a biotin-conjugated secondary antibody.
The fluorophore (if using fluorescent detection) may have been damaged by too much light exposure
  • Always keep fluorophore-conjugated secondary antibodies in the dark; exposure to too much light can lead to photobleaching.
Deparaffinization may be insufficient (if tissue is embedded in paraffin)
  • Deparaffinize sections longer and use fresh xylene.
Fixation procedures (if using formalin and paraformaldehyde fixatives) 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.
The antibody cannot penetrate the nucleus (if target protein is a nuclear protein)
  • Add a strong permeabilizing agent like Triton X to the blocking buffer and antibody dilution buffer. See our protocol on permeabilization techniques.
Permeabilization has damaged cell membranes (if target protein is a membrane protein)
  • Use a less stringent detergent (eg Tween 20 instead of Triton X). Or simply remove the permeabilizing agent from your buffers. See our protocol on permeabilization techniques.
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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High background

Browse the table below for possible causes of high background, and how to fix this — or prevent it altogether.


Possible causes

Solutions

The secondary antibody may be binding non-specifically
Blocking of non-specific binding might be insufficient
  • Increase the blocking incubation period and consider changing the blocking agent. We recommend 10% normal serum of the species of the secondary antibody for 1 hr.
Primary antibody concentration may be too high
Incubation temperature may be too high
  • Make sure you incubate sections at 4°C
Tissue not washed enough

Residual fixative or unbound antibodies remaining between steps can produce a false positive signal.

  • Wash the tissue extensively in buffer between all steps.
  • Increase the washing time.
Endogenous enzymes are active (if using enzyme-conjugated antibody)

Endogenous enzymes may give false positives, so you will need to block them with inhibitors prior to immunostaining.


Fixative is giving a fluorescent background signal (if using formalin / PFA fixatives and fluorescent detection)
  • Formalin/PFA usually fluoresce at green wavelengths, so try a fluorophore in the red or infared range to minimize overlap
Signal amplification may be too high (if using an amplification technique)
  • Reduce the amount of signal amplification (eg conjugate less biotin to secondary antibody if using biotinylation).
Too much substrate (if using enzyme-conjugated antibody)
  • Dilute the substrate and reduce substrate incubation time.
The tissue sections have dried out
  • Examine the tissue; sections with higher background staining at the edges than towards the centre are often dried out. 
  • Prevent tissue sections drying out by keeping them in a humidified chamber.

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