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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.
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Not enough antibody is bound to the protein |
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The antibody may not be suitable for IHC procedures which reveal the protein in its native state |
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Antibodies or amplification kits may have lost activity due to improper storage and handling |
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The protein of interest isn't present in the tissue |
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The protein of interest is present in low abundance |
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The fluorophore (if using fluorescent detection) may have been damaged by too much light exposure |
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Deparaffinization may be insufficient (if tissue is embedded in paraffin) |
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Fixation procedures (if using formalin and paraformaldehyde fixatives) may be masking the epitope |
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The antibody cannot penetrate the nucleus (if target protein is a nuclear protein) |
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Permeabilization has damaged cell membranes (if target protein is a membrane protein) |
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The buffer is contaminated with bacteria |
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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 |
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Blocking of non-specific binding might be insufficient |
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Primary antibody concentration may be too high |
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Incubation temperature may be too high |
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Tissue not washed enough | Residual fixative or unbound antibodies remaining between steps can produce a false positive signal.
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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.
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Fixative is giving a fluorescent background signal (if using formalin / PFA fixatives and fluorescent detection) |
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Signal amplification may be too high (if using an amplification technique) |
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Too much substrate (if using enzyme-conjugated antibody) |
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The tissue sections have dried out |
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