Immunohistochemistry Paraffin Troubleshooting

Symptom

Issue

Recommendations

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

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.

 

More Resources:

Immunohistochemistry Paraffin (IHC-P) Protocol