Staining in paraffin-embedded mouse cerebellum tissues by immunohistochemistry

Troubleshooting and using controls in IHC

Detailed troubleshooting tips and techniques for IHC.

Common problems

Using controls

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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Using Controls

It is essential to run controls in IHC staining experiments to confirm that the observed staining pattern is true, accurate and reliable.

Tissue controls

Positive tissue control

To validate the staining in your sample, use a positive control. This is a tissue section known to express the protein you are detecting.

How to finds a suitable positive tissue control

  • First, check the antibody datasheet. This will often provide a suggested positive control. Always ensure the tissue you use is from a tested species.
  • If the antibody datasheet does not list a positive control, we recommend the following:
  • Check to see if there are any Abreviews for the antibody. Any tissues, cells, or lysates that have been used successfully by these customers can be considered a suitable positive control.
  • Try looking at the UniProt/Swiss-Prot database links on the datasheet. These databases will often have a list of tissues that the protein is expressed in. These can also be considered suitable positive controls. 
  • If you still have difficulty finding a suitable control, we recommend performing a literature search on PubMed to see which tissues and cells express the protein of interest.

Negative tissue controls

Use a tissue sample known not to express the protein you are detecting. This is to check for non-specific binding and false-positive results.

Recommended negative control tissues are knockdown (KD) or knockout (KO) tissue samples.

Reagent controls

No primary controls

  • This is when the primary antibody is not added to the sample. This indicates if any non-specific binding or false positives may be due to non-specific binding of the secondary antibody.
  • The buffer containing no primary antibody is incubated with the same sample in the same way as usual.

Isotype controls

  • An isotype control is an antibody of the same isotype, clonality, conjugate, and host species as the primary antibody, that is raised against a molecule non-existent in the sample you are using. Usually, this is raised against a chemical or a non-mammalian protein.
  • Much like the no-primary control step, you would use this on your sample instead of the specific primary antibody. You would then use your secondary antibody as usual.
  • Use the same concentration for the isotype control antibody and the primary antibody. This will determine the level of background in your sample.

​Absorption controls

  • Tissue is incubated with primary antibody that has been pre-absorbed with a large molar excess the immunogen overnight. This removes antibodies that are expected to bind.
  • Little to no staining it expected; if you see a lot of staining, this indicates a lack of specifitiy.
  • Absorption controls are more reliable if the immunogen is a peptide, as antibody-protein immunogen mixtures may themselves cause high background staining in tissues due to non-specific interactions between the protein and the tissue. 

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