Immunohistochemistry Paraffin Troubleshooting



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Immunohistochemistry (IHC) handbook

IHC application resources

IHC sample preparation (frozen vs. paraffin-embedded)

IHC sample fixation (formalin vs. alcohol)

IHC blocking non-specific binding

IHC primary antibody selection

IHC epitope retrieval (HIER vs. PIER)

IHC detection methods

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TROUBLESHOOTING

The following troubleshooting guide is intended to explain causes and possible solutions for common problems observed in immunohistochemistry paraffin (IHC-P) staining.

No Signal

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 and secondary antibodies are compatible by checking the species reactivity.
  • Confirm the antibody can be used for assays in which the protein is in its native conformation.
  • Ensure that the secondary is working and compatible with your primary.

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.

Microscope Adjustments (Fluorescence)

  • Increase the exposure time of your camera.

 

Background

Antibody Concentration

  • Decrease the concentration of the primary/secondary antibody.

Blocking

  • The species of the blocking serum should be the same as the host of the secondary antibody e.g. use goat serum for blocking if your assay involves goat anti-mouse secondary.
  • Increase the incubation time or concentration of serum in the blocking buffer.

Antibody Application

  • Always incubate primary antibodies overnight at 4° C. Room temperature incubation increases unspecific binding and causes higher background.
  • Confirm that the secondary is not crossreacting 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

  • 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

Washing

  • Increase the amount of washes. Add very gentle agitation to the plates.

 

Sectioning Tissue

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.

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