Immunohistochemistry/Immunofluorescence Tips

IMMUNOHISTOCHEMISTRY / IMMUNOFLUORESCENCE TIPS

We are dedicated to helping our customers achieve exceptional results. While the information below can never be an exclusive solution to every problem you may encounter; it is our hope that you will find the information useful and even beneficial in troubleshooting any problems you may have. Of course, if you find you are still having problems you may submit them to us using this IHC-P Troubleshooting Form.

Lack of staining

Lack of antigen	Check protein expression by in situ hybridization.
Improper storage of antibodies	Follow storage instructions on the datasheet. Aliquot antibodies in a sufficent volume to make a working solution for a single experiment. Store aliquots in a manual defrost freezer (-20 to -70 °C) and avoid repeated freeze-thaw cycles.
Inactive primary or secondary antibodies	Check antibodies independently on a dot blot.
Inadequate tissue fixation	Increase or decrease fixation incubation, and try to change the fixative.
Over fixation	Reduce the duration of the immersion or post-fixation steps and be sure to include the Antigen Retrival step.
Antigen destroyed prior to staining	If quenching of endogenous peroxidase was done prior to the addition of primary antibodies, block peroxidase after incubation with the primary antibody.
Epitope altered during embedding or fixation	Embed tissue at 58 °C or below. Try restoring immunoreactivity through various antigen retrieval techniques.
Incomplete deparaffization	Use fresh xylene and increase xylene incubation time.
Insufficient antibody incubation	Incubate primary antibody overnight at 4°C
Photobleaching	Ensure to proceed with all steps in the dark.
Antigen retrieval was ineffective	Try to change the incubation time and also change solutions.
Antibodies are not 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.

High background

Hydrophobic interactions of the antibody and proteins in the tissue	Lower the salt concentration of the antibody stain buffer.
Tissue sections have dried out	Examine the tissue; sections with higher background staining at the edges than towards the center are often dried out. Prevent tissue sections drying out by keeping them in a humidified chamber.
Non-specific binding of primary antibody	Use the blocking step just prior to primary antibody incubation.
Non-specific binding of secondary antibody	Use an antibody that has cross-reactive IgG species removed (pre absorbed against sample species).
Ionic interactions	Ensure to change buffers.
Antibody is binding to Fc receptors on the target's cell surface	To block open binding sites, use 10% serum of the host secondary antibody.
Incubation temperature may be too high	Be sure to incubate at a temperature of 4°C.
Tissue inadequately washed	Include washing steps with fresh PBS/TBS.
Endogenous peroxidase	Use a peroxidase quenching buffer in blocking step before primary antibody.
Permeablization of membrane	Be sure to remove detergents from buffers.
Fixative was too strong	The epitope was modified, so be sure to change the antigen retrieval method.

Non-specific staining

Antibody incubation too long	Lessen the primary antibody incubation time.
Endogenous peroxidase	Use a peroxidase quenching buffer in the blocking step before the primary antibody.
Sections have dried-out	Ensure to keep slides in a humidity chamber.
Delay in fixation	Be sure to fix the tissue immediately.
Insufficient washing/blocking	Increase the washing time and number of washes.

Overstaining

Primary antibodies too concentrated

Dilute primary antibody solution. Perform a titration to determine optimal antibody dilution.

No or weak staining

Tissues have dried out

Cover the tissues in liquid at all times during the experiment.

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