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    IHC-emmbedded Tissue

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    iuventas
    Immunohistochemistry requires optimization of protocols for specific antibodies to avoid false positives and false negatives. Antigen retrieval may be needed to expose epitopes masked during tissue fixation and processing. Both primary and secondary antibodies can bind non-specifically, so controls are essential to validate specific staining. Attention to fixation, retrieval methods, blocking, and controls is crucial for reliable results.

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    IHC-emmbedded Tissue

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    iuventas
    19 views22 pages
    Immunohistochemistry requires optimization of protocols for specific antibodies to avoid false positives and false negatives. Antigen retrieval may be needed to expose epitopes masked during tissue fixation and processing. Both primary and secondary antibodies can bind non-specifically, so controls are essential to validate specific staining. Attention to fixation, retrieval methods, blocking, and controls is crucial for reliable results.

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    IHC-emmbedded tissue

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    Immunohistochemistry requires optimization of protocols for specific antibodies to avoid false positives and false negatives. Antigen retrieval may be needed to expose epitopes masked during tissue fixation and processing. Both primary and secondary antibodies can bind non-specifically, so controls are essential to validate specific staining. Attention to fixation, retrieval methods, blocking, and controls is crucial for reliable results.

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    19 views22 pages

    IHC-emmbedded Tissue

    Uploaded by

    iuventas
    Immunohistochemistry requires optimization of protocols for specific antibodies to avoid false positives and false negatives. Antigen retrieval may be needed to expose epitopes masked during tissue fixation and processing. Both primary and secondary antibodies can bind non-specifically, so controls are essential to validate specific staining. Attention to fixation, retrieval methods, blocking, and controls is crucial for reliable results.

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    of 22

    Overview of Immunohistochemistry

    (with a focus on wax-embedded sections)


    Overview of Immunohistochemistry
    (with a focus on wax-embedded sections)
    Overview of Immunohistochemistry
    “IHC is like cooking. There are many recipes out there,
    but some of them do not work out very well. However,
    when they do, they are great!
    You need the right ingredients, a dose of experience, a
    few tricks from old cooks, and a grain of common sense”.

    There are several traps to avoid….

    - sensitivity and specificity of the antibodies and


    technical procedure used are crucial to avoid false-
    positive and false-negative results.
    False-positive and false-negative results can be caused by:

     Primary antibody failing to detect their target antigen, even if it is present in


    the tissue
     Why?
     conformation changes induced by fixation or embedding
     low affinity of the antibody for the target
     failure of antibody to penetrate into tissue

     Antibodies binding non-specifically to other targets or tissue components


    (both primary and secondary antibodies)

    Blindly following an established protocol may prove insufficient. Each different


    antibody needs optimisation of the general protocol to ensure specific binding.
    FIXATIVES
    Influence of tissue preparation - fixatives
    Fixation changes the chemical properties of tissue constituents and alters 3D protein conformation
    by cross-linking. It has a major impact on affinity and selectivity of antibodies. Epitope masking can
    also occur when fixation alters penetration of antibodies into the tissue.

    European Journal of Neuroscience2008;28:2365-2370


    KO mouse -
    non-specific
    staining

    non-specific
    staining

    dependence of GABAA receptor 3 subunit-immunoreactivity on fixation

    European Journal of Neuroscience2008;28:2365-2370


    For fixatives consider:

    Reducing or enhancing fixation:


    Type of fixative (eg,formalin vs paraformaldehyde)
    pH
    Concentration of fixative (eg, 4% vs 10%)
    duration of fixation
    use of additives (eg, picric acid)

    Antigen retrieval
    ANTIGEN RETRIEVAL
    Antigen Retrieval
    • Epitope masking can occur when fixation alters penetration of antibodies into the tissue.
    • Antigen retrieval breaks down cross-links to expose the epitope and allow the primary antibody to bind.
    • Several retrieval methods exist, designed to break different types of cross-link.

    Eg: heating/boiling in acidic buffer


    enzyme digestion (eg, trypsin, Protease K)
    detergent (eg, triton, Tween20) Choose carefully, depending on nuclear or cytoplasmic staining
    repeated freeze/thaw

    Before retrieval After retrieval

    Beware false positive after Ag Retrieval


    The type of retrieval matters…

    (A) Formalin-fixed, paraffin-embedded


    osteosarcoma sample after CD31
    staining with standard heat
    induced epitope retrieval at 98°C

    (B) with optimized enzymatic epitope


    retrieval.

    (C) Formalin-fixed, paraffin-embedded


    osteosarcoma sample after
    FOXP3 staining with standard
    heat induced epitope retrieval at
    98°C

    (D) with optimized epitope retrieval at


    127°C.

    Kunz P, Fellenberg J, Moskovszky L, Sápi Z, et al. (2014) PLoS ONE 9(3): e90727. doi:10.1371/journal.pone.0090727
    The type of retrieval matters…
    ANTIBODY SPECIFICITY
    Antibody Specificity
    Monoclonal Antibodies: Polyclonal Antibodies:
    Primary Antibody Specificity: controls
    • Ideally, a tissue section should remain unstained after IHC processing if it is devoid of the target antigen.
    • In practice, this is generally not the case - IgGs bind with low affinity to numerous (mostly unidentified)
    tissue constituents.
    • Non-specific signals in tissues devoid of their target, such as a section from a knockout mouse, can also
    display non-specific staining.

    How to know staining is real?

    CONTROLS!

    1. Knockout mice
    2. Two antibodies raised against different epitopes of the antigen of interest (should show identical
    staining pattern)
    3. Inactivate antibody with its antigen prior to use (does not control for several targets sharing a
    common epitope recognised by the antibody)
    4. Positive control tissue - where staining pattern is known.

    Also:
    Use blocking solutions
    Optimise temperature, concentration and duration of incubation, as well as duration of rinsing steps.
    Secondary Antibody Specificity: Blocking background staining
    • Secondary antibodies are raised against IgGs of the species in which the primary antibodies were raised.
    • Used in fairly high concentration - non-specific binding to tissue components (eg, ECM) is higher than for
    primary Abs.

    IgG KO mouse

    Primary Ab =
    Primary Ab = migroglia
    migroglia marker
    marker Secondary Ab
    Secondary Ab - with
    - without blocking
    blocking

    • May also cross-react with IgGs from other species (relevant for dual staining).
    • Use highly adsorbed secondary antibodies.
    • Use blocking solutions.
    • Optimise temperature, concentration and duration of incubation, as well as duration of rinsing steps.
    • Use controls: No primary antibody
    Secondary Antibody Specificity:
    Blocking non-specific binding
    ENDOGENOUS PEROXIDASE
    False positive: Endogenous peroxidase
    Remember:
    Assess for false positives:

    • Does the tissue autofluoresce?


    • Are there endogenous peroxidases?
    • Do the secondary antibodies alone produce staining?
    • Does the primary antibody label the expected structures?

    Assess for false negatives:


    Failure to detect an antigen does not mean the antibody doesn’t work.

    • Is antigenicity lost during fixing?


    • Does tissue processing have a deleterious effect? (try frozen sections)
    • Does antigen retrieval give positive staining?
    • Did you dilute the antibody too far in advance? (Abs stick to plastic!)
    • Did you add peroxide to DAB substrate?
    • Did you use the correct secondary?
    • Did you make up ABC at least 30 minutes prior to use?
    • Do you need to amplify the signal?

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