1. Check the possible causes and find out the solution!


    Possible Causes

    What You Can Do?

    Insufficient blocking

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

    Interference from endogenous enzymes

    Perform H2O2 or Levamisole quenching.

    Non-specific binding from primary antibodies

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

    Inadequate washing

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

    Non-specific binding from secondary antibodies

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

    Non-specific binding from chromogen

    Perform chromogen incubation only. Use other chromogen if necessary.

    Interference from secondary antibody in multicolor staining

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

    Autofluorescence issue

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

  2. Check the possible causes and find out the solution!


    Possible Causes

    What You Can Do?

    Antigen retrieval too harsh

    Optimize retrieval steps to give the best morphology.

    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.

    Sectioning issue

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

    Autolysis has occurred

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

  3. Check the possible causes and find out the solution!


    Possible Causes

    What You Can Do?

    Over fixation

    Reduce fixation time. Perform antien retrieval to unmask epitopes.

    Insufficient fixation

    Increase fixation time or try other fixative.

    Fixation process delayed

    Fix immediately as tissue is extracted.

    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.

    Primary antibodies not suitable for IHC

    Choose an IHC-validated primary antibodies.

    Wrong secondary antibody used

    Make sure that primary and secondary antibodies match one another.

    Low expression of protein in tissue samples

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

    Insufficient deparafinization

    Make sure that parafin is removed completely before staining.