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Browse the table below for possible causes of no staining, and how to fix this — or prevent it altogether.
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.
|Not enough antibody is bound to the protein
|The antibody may not be suitable for IHC procedures which reveal the protein in its native state
|Antibodies or amplification kits may have lost activity due to improper storage and handling
|The protein of interest isn't present in the tissue
|The protein of interest is present in low abundance
|The fluorophore (if using fluorescent detection) may have been damaged by too much light exposure
|Deparaffinization may be insufficient (if tissue is embedded in paraffin)
|Fixation procedures (if using formalin and paraformaldehyde fixatives) may be masking the epitope
|The antibody cannot penetrate the nucleus (if target protein is a nuclear protein)
|Permeabilization has damaged cell membranes (if target protein is a membrane protein)
|The buffer is contaminated with bacteria
Browse the table below for possible causes of high background, and how to fix this — or prevent it altogether.
|The secondary antibody may be binding non-specifically
|Blocking of non-specific binding might be insufficient
|Primary antibody concentration may be too high
|Incubation temperature may be too high
|Tissue not washed enough
Residual fixative or unbound antibodies remaining between steps can produce a false positive signal.
|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)
|Signal amplification may be too high (if using an amplification technique)
|Too much substrate (if using enzyme-conjugated antibody)
|The tissue sections have dried out