Troubleshooting of Immunohistochemistry (IHC)

Here Creative Biolabs provides the common immunohistochemistry (IHC) troubleshooting tips. Though the tips provided here cover many different problems you may encounter in IHC, we hope that you will find the information beneficial to you and useful as a reference guide. If you ever need more assistance with your experiments, please contact us for additional help. Protocol of Immunohistochemistry (IHC) is available for you.

Contents

No Signal
Background
Cell/Tissue Morphology Destroyed
Inappropriate Staining

No Signal

Possible Cause	Recommended Solution
Antibody application	Increase the concentration or incubation time of the primary or secondary antibody.
Tissue fixation	Over fixation can cause epitope masking. Decrease the time or concentration of the fixative. Under fixation can cause heavy edge staining with little to no positive signal in middle of your specimen.
Antibody compatibility	Confirm that your primary-secondary antibodies are compatible. Confirm the antibody can be used for assays in which the protein is in its native conformation.
Permeabilization	Use 0.5-1.0% Triton detergent in the buffers in order to allow full permeabilization of antibody and buffers into the tissue sections.

Background

Possible Cause	Recommended Solution
Antibody concentration	Titer antibody to determine optimal concentration needed to promote a specific reaction of the primary and the secondary antibodies.
Hydrophobic interactions of the antibody and proteins in the tissue	Lower the ionic strength of the antibody diluent (particularly, monoclonal antibodies respond well to reducing the salt concentration).
Blocking	The species of the blocking serum should be the same as the host of the secondary antibody. Increase the incubation time or concentration of serum in the blocking buffer.
Non-specific binding of primary and/or secondary reagents to tissues	Use blocking step just prior to primary antibody incubation.
Non-specific binding of secondary antibody	Use an antibody that has had cross-reactive IgG species removed (absorbed against sample species)
Antibody application	Better to incubate primary antibodies overnight at 4°C. RT incubation increases non-specific binding and causes higher background. Confirm that the secondary is not cross-reacting with the cells by performing the assay without the primary.
DAB reaction	Do not overexpose the DAB reaction. Rinse DAB off slides sooner. Endogenous peroxidases are activating the reaction. Quench with hydrogen peroxide.
ABC method	Endogenous biotin is activating the complex. Block with avidin/biotin blocking kit.
Cells drying (Fluorescence)	Fluorescent signal will be lost if the cells are allowed to dry. Ring coverslips with nail polish.
Microscope adjustments (Fluorescence)	Increase the exposure time of your camera.
Spectral overlap (Fluorescence)	If double or triple labeling the cells, confirm that the secondaries do not overlap into the same spectral range.
Inadequate washing	Increase the amount of washes. Add very gentle agitation to the plates.
Ionic interactions	Increase the ionic strength of the diluent buffer.
Reagents sticking to old or poorly prepared slides.	Start over with freshly prepared or purchased slides.

Cell/Tissue Morphology Destroyed

Possible Cause	Recommended Solution
Holes in the tissue	Ensure the blade is adequately sharp. Adjust the cutting speed. Perfuse at a lower rate.
Tissue falling off Slides	Use coated slides. Place slides in covered dish with a small amount of 16% formaldehyde on the bottom to fix the tissues to the slides. Be gentle when using an antigen retrieval method and avoid heavy agitation of the slides.
Tissue curling	Decrease the temperature of the cryostat or open cryostat lid more. Cut thicker sections.
Antigen retrieval methods are too harsh	Empirically determine the conditions that preserve tissue morphology while restoring the immunoreactivity of the antigen.
Poor resolution of tissue morphology	Cut thinner tissue sections. Ice crystals may have destroyed morphology of frozen sections.
Under fixation has physically damaged the tissue or cells during the staining process.	Increase fixation time and/or add a post-fixation step. Increase the fixative/tissue ratio. Cut smaller pieces of tissue for more thorough immersion-fixation.
Autolysis of tissue leading to staining of necrotic debris.	Increase the fixation time, ratio. Consider using cross-linking fixative.

Inappropriate Staining

Possible Cause	Recommended Solution
Fixation method is inappropriate for the target	Try a different fixative or increase the fixation time.
Antigen retrieval may be inappropriate for this antigen or tissue.	Try different antigen retrieval method.
Electrostatic charge of the antigen has been altered	Try adjusting the pH or cation concentration of the antibody diluent.
Delay in fixation caused diffusion of the antigen.	Fix tissue promptly. Try a cross-linking fixative rather than organic (alcohol) fixative.