The following guide serves as a checklist for the possible causes and solutions with respect to some of the most commonly encountered problems from the IHC assays.

  1. No or Weak Staining

    Possible Cause Solution
    1 Slides lose signal over time during storage Prepare slides with freshly-sliced tissues
    Store slides at 4°C
    Do not bake slides before storage
    2 The antibody used is not suitable for IHC procedures which detect proteins in its native conformation Check the antibody datasheet to make certain that it has been validated for IHC applications
    Check the antibody is applicable to the right IHC samples (paraffin sections vs. frozen samples)
    Perform Western blot in both its native and denatured forms to ensure that the antibody detects the native form
    3 Fixation procedures (using formalin and paraformaldehyde fixatives) have masked the epitope that the antibody recognizes Use different antigen retrieval methods to unmask the epitope (HIER or PIER)
    Fix the sections in a shorter time
    4 The primary and/or secondary antibody have lost its activity due to improper storage, dilution or excessive freezing and thawing Run positive controls to ensure that the primary and/or secondary antibody is working properly
    Store the antibodies according to manufacturer instructions
    Avoid contamination and light on antibodies
    5 Insufficient deparaffinization Increase the deparaffinization time
    Use fresh dimethylbenzene
    6 The protein is located in the nucleus and the antibody cannot penetrate the nucleus Add a permeabilizing agent (e.g. Triton X) to the blocking buffer and antibody dilution buffer
    7 The PBS buffer has contaminated with bacteria that damage the phosphate groups on the protein of interest Add 0.01% azide in the PBS antibody storage buffer
    Use fresh sterile PBS
    8 The primary antibody and the secondary antibody are not compatible Use a secondary antibody that was raised against the species in which the primary was raised (e.g. if the primary antibody was raised in mouse, an anti-mouse secondary antibody should be used)
    Check that the isotypes of the primary and secondary are compatible
    9 The protein is not present in the tissue of interest or has not sufficiently expressed Run positive controls to ensure that target protein is present in the tissue
    Include an amplification step in your protocol
    Use higher antibody concentration
    10 Insufficient antibody to detect protein of interest Use a higher antibody concentration
    Incubate for a longer time (e.g. overnight) at 4°C
    11 Tissues dry out Cover the tissues in liquid at all time during the experiment
  2. High Background

    Possible Cause Solution
    1 The blocking serum is incorrect Make sure to block according to the provided protocol
    2 Blocking is insufficient
    (Do not over-block the tissue because antigenic sites may be masked)
    Increase blocking incubation period
    Change blocking reagent:
    (a) For sections: 10% normal serum (1 hr)
    (b) For cell cultures: 1-5% BSA (0.5 hr)
    3 The primary antibody concentration is too high Titrate the antibody to determine the optimal concentration
    Incubate at 4°C
    4 Non-specific binding by secondary antibody Run a secondary control without primary antibody: If you see staining with your secondary only:
    (a) Change your secondary antibody or
    (b) Use secondary antibody that has been pre-adsorbed against the immunoglobulin of the species from which your samples were obtained
    Block your sample with serum from the same species as the host in which the secondary antibody was raised
    5 Endogenous peroxide or phosphatase is active Quench the endogenous peroxidase or phosphatase activity by enzyme inhibitors:
    (a) Peroxidase: H2O2 and methanol (v/v: 0.3%:99.7%)
    (b) Phosphatase: 2 mM Levamisol
    6 Too much amplification (Refer to solution #9 from no/weak staining) Reduce amplification incubation time
    Dilute the secondary antibody
    7 Too much substrate was applied (enzymatic detection) Further dilute substrate
    Reduce substrate incubation time
    Choose substrate of higher S/N ratio e.g. Metal-enhanced DAB
    8 Tissue section is not thin enough for reagent penetration Prepare thinner section
    9 Incubation temperature is too high Incubate samples at 4°C
    10 Primary antibody was raised in the same species as source of tissue (therefore, secondary antibody recognizes and binds to everywhere on the entire tissue because it was raised against that species) Use primary antibody raised against a species which is different from the source of tissue
    Use biotinylated primary antibody and conjugated streptavidin for the detection system
    11 Secondary antibody binds endogenous IgG Include control slide stained without the primary antibody to confirm whether the secondary antibody is the source of the background
    12 Fixation reagents are still present (Due to insufficient tissue washing) Wash the tissues extensively with PBS buffer
    13 Reaction between chromogens and PBS buffer in tissue or cell samples Before incubating with the substrate, use Tris buffer to wash the samples
    14 Membrane damage by permeabilization  Use a less stringent detergent such as Tween 20 (instead of Triton X)
    Remove permeabilizing agent from your buffers
    15 Insufficient deparaffinization Increase the deparaffinization time
    Use fresh dimethylbenzene
    16 High levels of endogenous biotin in biotin-based detection systems for samples (e.g. liver and kidney tissues) Perform biotin block after normal blocking procedure (before primary antibody incubation)
    Use polymer-based detection
    17 Use of polyclonal primary antibody Use monoclonal primary antibody to reduce cross-reactivity