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.

SYMPTOMS

> Lack of staining
> High background
> Non-specific staining
> Overstaining
> No or weak staining


LACK OF STAINING
ISSUERECOMMENDATION
Lack of antigenCheck protein expression by in situ hybridization.

Improper storage of antibodiesFollow 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 antibodiesCheck antibodies independently on a dot blot.

Inadequate tissue fixationIncrease or decrease fixation incubation, and try to change the fixative.

Over fixationReduce the duration of the immersion or post-fixation steps and be sure to include the Antigen Retrival step.

Antigen destroyed prior to stainingIf 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 fixationEmbed tissue at 58 °C or below. Try restoring immunoreactivity through various antigen retrieval techniques.

Incomplete deparaffizationUse fresh xylene and increase xylene incubation time.

Insufficient antibody incubationIncubate primary antibody overnight at 4°C.

Photobleaching Ensure to proceed with all steps in the dark.

Antigen retrieval was ineffectiveTry to change the incubation time and also change solutions.

Antibodies are not compatibleMake 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
ISSUERECOMMENDATION
Hydrophobic interactions of the antibody and proteins in the tissue Lower the salt concentration of the antibody stain buffer.

Tissue sections have dried outExamine 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 interactionsEnsure to change buffers.

Antibody is binding to Fc receptors on the target's cell surfaceTo block open binding sites, use 10% serum of the host secondary antibody.

Incubation temperature may be too highBe sure to incubate at a temperature of 4°C.

Tissue inadequately washedInclude washing steps with fresh PBS/TBS.

Endogenous peroxidaseUse a peroxidase quenching buffer in blocking step before primary antibody.

Permeablization of membraneBe 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
ISSUERECOMMENDATION
Antibody incubation too longLessen the primary antibody incubation time.

Endogenous peroxidaseUse a peroxidase quenching buffer in the blocking step before the primary antibody.

Sections have dried-outEnsure to keep slides in a humidity chamber.

Delay in fixationBe sure to fix the tissue immediately.

Insufficient washing/blockingIncrease the washing time and number of washes.


OVERSTAINING
ISSUERECOMMENDATION
Primary antibodies too concentratedDilute primary antibody solution. Perform a titration to determine optimal antibody dilution.


NO OR WEAK STAINING
ISSUERECOMMENDATION
Tissues dried outCover the tissues in liquid at all times during the experiment.