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  • IHC training
    Part 1: Basics


    Welcome to our training series on immunohistochemistry (IHC). We begin with essentials like sample preparation and antibody selection and then guide you through immunostaining protocols and troubleshooting.

    In Part 1 of IHC training, we’ll show you how to get the best possible start. How well you prepare your sample is going to be a significant determinant in how good your data are. Here, we take you through the main steps involved in the optimal sample preparation and processing for IHC.


    Part 1 overview

    1.1 What is immunohistochemistry?
    1.2 Specimen formats
    1.3 Fixation
    1.4 Embedding and sectioning


    1.1 What is immunohistochemistry?

    Immunohistochemistry (IHC) is a method to access the distribution and localization of antigens in tissue sections with the use of antibody-antigen interactions.

    IHC is often used to diagnose tissue abnormalities in diseases such as cancer. In general, IHC provides valuable perspective and support that can contextualize data obtained from other methods.

    An IHC stain relies on antibodies that recognize the target antigen. You can use either chromogenic or fluorescent-based detection systems to visualize this antibody-antigen interaction. In chromogenic detection, an antibody is conjugated to an enzyme that produces a colored precipitate when exposed to a chromogen. In fluorescent detection, an antibody is conjugated to a fluorophore.

    This short video will introduce you to the main principles of IHC.


    1.2 Sample preparation

    Sample preparation is a key to producing high-quality staining during IHC. Sample preparation includes processes such as fixation, embedding, and sectioning.

    The two main methods of preserving tissues for IHC are paraffin embedding of formalin-fixed tissues and freezing. In this video, you’ll learn the differences between these two methods and their advantages and limitations.


    See below a quick guide on the main differences between paraffin-embedded and frozen sections for IHC.

    IHC – Paraffin-embedded IHC - Frozen
    Section thickness 3–4 µm 4–10 µm
    Fixation Most commonly used - 10% neutral-buffered formalin (NBF) Paraformaldehyde, methanol, ethanol, or acetone
    Pre-embedding Pre or post-sectioning
    Sectioning Microtome Cryostat
    Storage Multiple years at room temperature (antigen may change over time) 1 year at -80°C (longer at -190°C).
    Advantages Optimal balance between preservation of morphology and antigenicity Antigens are in a more 'native' state
    Very stable and easy to work with Ideal for antigens that don't tolerate standard aldehyde fixation and paraffin processing
    Used for phosphorylated epitopes
    Disadvantages Overfixation (longer than 24 hours) can mask the epitope Morphology can be poorer compared to paraffin if ice crystals form
    Requires de-waxing before immunostaining Thicker sections may give lower resolution and poorer images
    Requires antigen retrieval if fixed with a crosslinking agent (aldehyde) Samples are more fragile


    1.3 Fixation

    Fixation immobilizes antigens while retaining cellular and subcellular structure. Fixation prevents the autolysis and necrosis of excised tissues, preserves antigenicity, and helps to preserve cellular elements during tissue processing.

    The fixation method used will depend on the sensitivity of the epitope and may require some optimization. In this video, you’ll learn more about standard fixation methods in IHC.

    Standardized fixatives for each type of antigen are essential for reproducible staining - an antigen that has been inappropriately fixed may not be detected.

    Here are our recommendations on choosing a fixative:

    • Most proteins, peptides, and enzymes of low molecular weight: for cells/cytological preparations, use 4% formaldehyde; for tissue sections, use 10% neutral-buffered formalin (NBF)
    • Delicate tissue: Bouin's fixative
    • Small molecules such as amino acids: 4% formaldehyde
    • Blood-forming organs (eg liver, spleen, bone marrow): Zenker’s solution
    • Connective tissue: Helly's solution
    • Nucleic acids: Carnoy’s solution
    • Large protein antigens (eg immunoglobulin): ice-cold acetone or methanol (100%)
    • Nuclear morphology: zinc formalin
    • For electron microscopy: 4% formaldehyde, 1% glutaraldehyde

    Get the fixation protocol.


    1.4 Embedding and sectioning

    Once you’ve fixed your tissue, you need to process it so that it is adequately supported for cutting into sections of usually 4–10 µm thickness. Tissues are fixed with aldehydes and then dehydrated, cleared, and infiltrated with paraffin, which is the medium enabling tissue handling and subsequent sectioning. Alternatively, unfixed tissues can be snap frozen in liquid nitrogen and embedded in a medium such as OCT (optimal cutting temperature compound). In this case, a short fixation will occur before tissue sectioning. The following protocol covers:

    • Paraffin tissue processing
    • Frozen tissue embedding
    • Glycol methacrylate (GMA) embedding

    Get the tissue processing protocol.

    This video guides you through both embedding and sectioning for paraffin sections.

    Sectioning paraffin-embedded tissue video protocol


    Summary

    Hopefully, you now have a great foundation from which to start planning your IHC experiments. You should have a good understanding of sample preparation, specifically:

    • The two main methods of preserving tissues for IHC
    • Tissue fixation and embedding methods

    In Part 2, we’ll cover the immunostaining protocol’s steps such as antigen retrieval, permeabilization, blocking, selection of primary and secondary antibodies and detection systems.

    Start Part 2 now!