Possible Causes

What You Can Do?

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Insufficient blocking

Select appropriate serum as blocking buffer. Blocking for 1 hour at room temperature.

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Interference from endogenous enzymes

Perform H2O2 or Levamisole quenching.

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Non-specific binding from primary antibodies

Dilute primary or secondary antibody. Choose another IHC-validated primary antibodies.

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Inadequate washing

Increase washing cycles/time. Increase salt/detergent concentration
for stronger washes.

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Non-specific binding from secondary antibodies

Perform secondary antibody incubation only. Use pre-adsorbed secondary antibody.

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Non-specific binding from chromogen

Perform chromogen incubation only. Use other chromogen if necessary.

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Interference from secondary antibody in multicolor staining

Make sure that the fluorochrome does not overlap with one another.

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Autofluorescence issue

Make sure that there is no endogenous background caused by tissue itself. Check under fluorescence microscope prior to staining to identify autofluorescence.

Possible Causes

What You Can Do?

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Antigen retrieval too harsh

Optimize retrieval steps to give the best morphology.

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Tissue sections peeled off slide

Dry samples for 2-4 hours at 60°C.
Tissue with high lipid content (eg breast tissues) should be dried for longer time.

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Sectioning issue

Cut thinner slides for better resolution: 3-5 μm. Use a new/sharper blade.

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Autolysis has occurred

Fix samples as soon as possible.
Choose other fixatives to accelerate penetration.
Fixative perfusion might be necessary for larger tissues.

Possible Causes

What You Can Do?

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Over fixation

Reduce fixation time. Perform antien retrieval to unmask epitopes.

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Insufficient fixation

Increase fixation time or try other fixative.

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Fixation process delayed

Fix immediately as tissue is extracted.

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Permeabilization issue

For nuclear/cytoplasmic proteins, add permeabilization agent (eg Triton X-100, Saponin) in blocking and antibody incubation buffer. 
For membrane/tight junction proteins, avoid permeabilization agent.

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Primary antibodies not suitable for IHC

Choose an IHC-validated primary antibodies.

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Wrong secondary antibody used

Make sure that primary and secondary antibodies match one another.

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Low expression of protein in tissue samples

Use signal amplification methods (eg: HRP Polymer ARG80982, ARG80967, ARG80966)

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Insufficient deparafinization

Make sure that parafin is removed completely before staining.