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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|
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
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.