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Trouble-free IHC with optimized kits and reagents
For IHC, to ensure that you get the data you need on your first experiment, we recommend our optimized kits and reagents:
Alternatively, you can try the tips below:
The primary antibody and the secondary antibody are not compatible
Not enough primary antibody is bound to the protein of interest
The antibody may not be suitable for IHC procedures which reveal the protein in its native (3D form)
The primary/secondary antibody/amplification kit may have lost its activity due to improper storage, improper dilution or extensive freezing/thawing
The protein is not present in the tissue of interest
The protein of interest is not abundantly present in the tissue
The secondary antibody was not stored in the dark (if your detection system is immunofluorescence).
Deparaffinization may be insufficient
Fixation procedures (using formalin and paraformaldehyde fixatives) may be modifying the epitope the antibody recognizes
The protein is located in the nucleus and the antibody (nuclear protein) cannot penetrate the nucleus
The PBS buffer is contaminated with bacteria that damage the phosphate groups on the protein of interest
Blocking of non-specific binding might be absent or insufficient
The primary antibody concentration may be too high
Incubation temperature may be too high
The secondary antibody may be binding non-specifically
Tissue not washed enough, fixative still present
Endogenous peroxidases are active
Fixation procedures (using formalin or paraformaldehyde fixatives) are causing autofluorescence (if your detection system is immunofluorescence)
Too much amplification (amplification technique)
Too much substrate was applied (enzymatic detection)
The chromogen reacts with the PBS present in the cells/tissue (enzymatic detection)
Permeabilization has damaged the membrane and removed the membrane protein (membrane protein detection)
Primary/secondary antibody concentration may be too high
Endogenous peroxidases are active
The primary antibody is raised against the same species as the tissue stained (e.g. mouse primary antibody tested on mouse tissue). When the secondary antibody is applied it binds to all the tissue as it is raised against that species
The sections/cells have dried out
Why positive controls are necessary
To validate the staining in your sample, use a positive control. This is a section from a cell line or tissue known to express the protein you are detecting. A positive result from the positive control, even if the samples are negative, will indicate the procedure is optimized and working. It will verify that any negative results are valid.
How to know what tissue type or cell line is a suitable positive control
Why negative controls are necessary
Use a section from a cell line or 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.
No primary controls
Controls for using a transfected cell line:
We recommend including an endogenous control if you are testing a sample of recombinant protein. This should be an essential part of the experimental plans.