IHC Technical Resources

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Introduction

Immunohistochemistry (IHC) is a method for detecting antigens or haptens in cells of a tissue section by exploiting the principle of antibodies binding specifically to antigens in biological tissues. The antibody-antigen binding can be visualized in different manners. Enzymes, such as Horseradish Peroxidase (HRP) or Alkaline Phosphatase (AP), are commonly used to catalyze a color-producing reaction.

IHC is widely used in many research and clinical laboratories because this technique makes it possible to visualize the distribution and localization of specific cellular components within cells and in proper tissue context. There are numerous IHC methods that can be used to localize antigens. The method selected should include consideration of parameters such as the specimen types and assay sensitivity.

In this article, the following topics will be covered:

  1. Sample Preparation
  2. Fixation
  3. Tissue Sectioning
  4. Paraffin Embedding
  5. Inactivation and Blocking
  6. Antigen Retrieval
  7. Detection
  8. Chromogens, Counterstains and Mounting Media
  1. Sample Preparation

    Sample collection and preparation play an important role in IHC as the antigen exhibition and location are largely depend on the quality of tissue sample.

    1. Cell Sample

      1. Adherent cells
        • Cell Climbing: Grow adherent cells on multi-aperture culture plates with coverslip, culture vessels or chamber slide
        • Direct Cell Culture: Culture adherent cells directly on culture vessels or multi-aperture culture plates
      2. Non-Adherent Cells
          Cell Smear: Adhere non-adherent cells on coverslip with chemical bond
        • Eccentric Cell Smears: Adhere non-adherent cells on culture vessels by cell micro-centrifuge
    2. Tissue Sample

      Tissue samples are typically taken from specimens of various sources: biopsy, surgery, animal model and autopsy. The first three types of specimens give fresh tissues while the last one (autopsy) is taken after an animal has died for two hours which is more or less a postmortem autolysis. As antigens may denature, disappear and diffuse, autopsy specimen should be fixated as soon as possible so as not to influence its label.

      Exercise caution when collecting, fixating and sectioning the samples

      • Use sharp knife and scissors to avoid extrusion damage
      • Cutter should be flat, small and thin (Normal size is 1.0 cm × 1.0 cm × 0.2 cm)
      • Eliminate fat tissue and calcification zone
      • Collect samples from live animals and fix samples immediately after wash
      • Choose diseased instead of necrotic region
      • Choose normal tissue as control if necessary
      • Make paraffin-embedded tissue or frozen tissue immediately after sectioning or store the tissues in liquid nitrogen container or refrigerator at -70℃
  2. Fixation

    1. Purposes

      • Keep cell sharp and tissue shape to prevent postmortem autolysis, putridness, endogenic and exogenic enzyme activity
      • Maintain cell structure and position by preventing antigen diffusion through transfer of protein, fat, sugar and enzymes of cell into insoluble substances
      • Precipitate and curdle materials in tissue to produce different refraction
      • Indurate tissues to enhance working with glass slides
      • Prevent cell from shrinking and swelling
      • Give color to clarify tissues by different affinity to coloring agent
    2. Selection of Fixing Solution

      Below is a list of commonly used fixing solutions. You may need to test whether a specific type of solution is appropriate for your detected antigens because there is no standard fixing solution for different kinds of antigen immobilization.

      1. Acetone and Alcohol

        These two types of solutions, which are primary fixing solutions, play a role of precipitating sugars and fat as well as maintain the immunologic competence.

        • Alcohol is ineffective to maintain low molecular weight protein, polypeptide and cytoplasmic proteins. However, it can be mixed with glacial acetic acid, ethyl ether, chloroform and formaldehyde.
        • Acetone is often used for frozen tissue and cytological smears because it has a strong penetrability and dehydration property.
      2. Aldehyde

        It is a di-functional cross-linking agent which is widely used due to its strong penetrability, low contractibility and low background. It helps keep the cross-linking between tissues and maintain antigen.

        • Formalin (10% neutral buffered) is the most widely used
        • 4% paraformaldehyde is better than formaldehyde
        • Bouin’s solution (containing picric acid) is the most widely used in histology and pathology
        • Zamboni’s solution is applied to light and electron microscopic immunocytochemistry and is better than formaldehyde in ultrastructural organization maintenance
      3. Non-Aldehyde
        • Carbodiimide, dimethylacetamide, dimethyl-suberimidate, para-benzoquinone are widely used in tissue fixation of peptide hormones.
        • These fixation agents are better mixed with glutaric dialdehyde or paraformaldehyde.
      4. In recent years, a new type of formaldehyde-free fixing solution has become available. With low toxicity and degradable chemical agent, this solution has gained a broad popularity in IHC, regular pathological examinations and molecular pathology detections due to the use of non-protein cross linking, strong DNA/RNA preservation, and absence of cell vacuole, tissue shrinkage and pyknosis.

    3. Method and Time

      1. Method: Immersion

        The immersion method marinates the tissue in fixing solution (at 4℃ if needed) for a specified period which is determined by the antigen stability and type of fixing solution used. Biopsy and surgical specimens as well as other non-irrigation tissues commonly employ this fixation method.

      2. Method: Irrigation

        This method has the ability to fix tissues fully and quickly, suppressing the interference of endogenous peroxidase. Therefore, it is a method of choice in animal experiments.

      3. Fixation Time

        The fixation time depends on the tissue thickness, solution concentration and experimental temperature. In principle, the time is directly proportional to the tissue thickness but inversely proportional to the solution concentration.

      4. Exercise caution when fixating tissues

        • Do not over-fix the tissues
        • Keep the tissues fresh after fixation
        • Use enough fixing solution and wash it off completely after fixation
        • Use tissues of size less than 2 cm × 1.5 cm × 0.3 cm (Thickness < 0.3 cm)
  3. Tissue Sectioning

    1. Slide Pre-Treatment

      The following pre-treatment procedure is designed to prevent peeling caused by elevated temperature, high pressure, radiation and other factors.

      1. Microscopic Slide

        Due to the oil attached on surface, a new slide should be dipped into cleaning solution for 12 to 24 hours. After washing the slide more than 5X in distilled water, dip it into 95% alcohol for 2 hours followed by drying with simple wiping or in an infrared oven. Pay attention to avoid scratching the slide during washing.

        Note: Microscopic slide for IHC is required to be 5 μm. However, the slide for nervous tissue should be 20-100 μm to enhance the tracking of never fiber direction.

      2. Coverslip

        The coverslip pre-treatment procedure is very similar to the one for the microscopic slide except that dipping and cleaning should be completed in 2 hours because the coverslip is much thinner.

      3. Tissue Section Mounting
        • Dilute 3-Amino Propyl Tri-ethoxy Silane (APES) by acetone
        • Coat the microscopic slide with APES
        • Apply tissue on the coated slide followed by adding a drop of mounting medium (Glycerol Gelatin) on the tissue
        • Hold the coverslip at 45°, allowing the drop to spread along the edge of the slip
        • Slowly cover the tissue entirely with the coverslip
        • Incubate the slide at 80℃ for 1 hour if there is an adhering problem with a tissue section
    2. Tissue Section Types

      1. Frozen

        The most important feature for this type of tissue section is to keep antigen’s immune-competence completely, especially for the cell surface antigen. Both fresh and fixed tissues can be processed as frozen tissues. However, the tissues must be dried (or primary fixed) and stored at low temperature.

      2. Paraffin-Embedded

        Paraffin-embedded tissue section is normally sliced by a rotary microtome to give a thickness of 2-7 μm. With proper treatment, the section reveals clear tissue structure and exact antigen location to enable high medical-value pathology researches and retrospective studies. This section type can be stored at 4℃ for long term use.

    3. Processing

      1. Cell on Coverslip
        • Place settled coverslip in culture bottle or perforated plate
        • Take out coverslip after cell growth has reached 60%
        • Fix coverslip with cold acetone or 4% paraformaldehyde for 10 to 30 min
        • Store cell (in gelatin) at -20℃
      2. Cell Smear
        • Collect non-adherent cells and wash 2X by cold PBS buffer
        • Re-suspend cells with PBS buffer
        • Add 30-50 µL to settled slide and smear it evenly
        • Air dry slide a little bit and cover cell with 4% paraformaldehyde for 2-4 hours
        • Store cell (in gelatin) at -20℃
  4. Paraffin Embedding

    Five major steps are involved in paraffin embedding: fixation, dehydration, transparentizing, immersion and embedding.

    1. Fixation

      Please refer to the Fixation section described above.

      1. Fixation

        Please refer to the Fixation section described above.

      2. Dehydration

        This step removes water completely, creates a condition for the next step and hardens the tissue of interest. The dehydrating agents describes below are completely miscible with water and can be prepared in different volumetric ratios with water.

        1. Ethanol

          As the most commonly used dehydrating agent, ethanol has a strong water separation and tissue hardening capability. However, since ethanol has strong penetration and contractility, its concentration should be progressively increased to avoid tissue excessive shrinking.

        2. Acetone

          As a usual substitute for alcohol, acetone acts as both a fixing solution and fast dehydrating agent. Pay attention to the dehydrating time with acetone as it tends to over harden tissues.

      3. Transparentizing

        After dehydration, the tissue of interest requires a transparentizing step because the dehydrating agent used in the previous step is immiscible with the paraffin from one of the subsequent steps. The addition of transparent reagent helps paraffin absorb into the tissue. Common transparent reagents are:

        1. Xylene

          As the most widely used transparent reagent, xylene is miscible with both ethanol and acetone, and it acts as a fusing agent for paraffin wax. Since xylene has a strong and fast contractility to tissue, the tissue should not be immersed for an extended time period or it will be over crisp and too hard.

        2. Benzene and Toluene

          These reagents are similar to xylene. However, they have weak and slow contractility to tissue, and therefore the tissue can be immersed in these reagents for a longer time. Note that benzene and toluene have high toxicity and must be handled with care.

        3. Chloroform

          Compared to xylene, benzene and toluene, chloroform is a much gentler reagent. However, it has a small refractive index, and the tissue should thus be immersed in chloroform for a longer time than the other transparent agents in order to guarantee complete penetration.

        4. Cedar Oil

          Due to minimal sclerification created by cedar oil, it is an appropriate transparent agent for fine and soft tissues. Super hard tissues (e.g. skin tissue) and dense fibrous tissue are also easier to be sectioned after immersing in cedar oil. However, this oil is not useful for other common tissue sections because of its high concentration and weak penetrability.

        In recent years, a new type of environment-friendly transparent agent has appeared in market. Instead of aromatic compound, the main components of this new reagent is alkanes, and it can be used to replace xylene.

      4. Immersion

        After transparentizing, the tissue can be immersed in molten paraffin wax so that it adsorbs the wax-substituting transparent agent. Based upon the melting point of wax, immersion should be performed at 54-64℃.

      5. Embedding

        This is a process of treating the tissue in a paraffin box so that the paraffin wax cools down and solidifies. The treatment conditions (using ethanol and xylene as an example) are shown in the table below. After cooling is completed, the tissue will be ready for sectioning and suitable for storage.

        Step Reagent Time (Hours)
        1 75% Ethanol 0.5 to 2
        2 85% Ethanol 0.5 to 2
        3 95% Ethanol 2
        4 95% Ethanol 2
        5 95% Ethanol 2
        6 100% Ethanol 0.5 to 1
        7 100% Ethanol 0.5 to 1
        8 100% Ethanol 0.5 to 1
        9 Xylene 0.25
        10 Xylene 0.25
        11 Xylene 0.25
        12 Paraffin Wax 0.5
        13 Paraffin Wax 1 to 2
        14 Paraffin Wax 1 to 2
    2. Tissue Section Types

      1. Frozen

        The most important feature for this type of tissue section is to keep antigen’s immune-competence completely, especially for the cell surface antigen. Both fresh and fixed tissues can be processed as frozen tissues. However, the tissues must be dried (or primary fixed) and stored at low temperature.

      2. Paraffin-Embedded

        Paraffin-embedded tissue section is normally sliced by a rotary microtome to give a thickness of 2-7 μm. With proper treatment, the section reveals clear tissue structure and exact antigen location to enable high medical-value pathology researches and retrospective studies. This section type can be stored at 4℃ for long term use.

    3. Processing

      1. Cell on Coverslip
        • Place settled coverslip in culture bottle or perforated plate
        • Take out coverslip after cell growth has reached 60%
        • Fix coverslip with cold acetone or 4% paraformaldehyde for 10 to 30 min
        • Store cell (in gelatin) at -20℃
      2. Cell Smear
        • Collect non-adherent cells and wash 2X by cold PBS buffer
        • Re-suspend cells with PBS buffer
        • Add 30-50 µL to settled slide and smear it evenly
        • Air dry slide a little bit and cover cell with 4% paraformaldehyde for 2-4 hours
        • Store cell (in gelatin) at -20℃
  5. Inactivation and Blocking

    1. Inactivation

      When either the horseradish peroxidase (HRP) or alkaline-phosphatase (AP) system is applied for IHC, activation of endogenous enzymes should be blocked or inhibited to avoid producing non-specific binding.

      1. Endogenous HRP Inactivation
        • Incubate the paraffin embedded section in 3% H2O2 for 10 min
        • Incubate the frozen section or cell section in solution composed of methanol and 3% H2O2 (v/v: 4:1) for 30 min
      2. Endogenous AP Inactivation
        • Incubate the sample section in 0.1 mM Levamisole
        • Note: Levamisole cannot inhibit the AP activation of endogenous intestine tissue
      3. Blocking

        Residual sites on the tissue section may bind to secondary antibody and produce follow-up false positive results. Therefore, serum from the same species as the secondary antibody is commonly used for blocking. Animal’s autoantibody in the serum can bind to the sites in advance. Blocking should be done at room temperature for 10-30 min (avoid excessive blocking).

    2. Tissue Section Types

      1. Frozen

        The most important feature for this type of tissue section is to keep antigen’s immune-competence completely, especially for the cell surface antigen. Both fresh and fixed tissues can be processed as frozen tissues. However, the tissues must be dried (or primary fixed) and stored at low temperature.

      2. Paraffin-Embedded

        Paraffin-embedded tissue section is normally sliced by a rotary microtome to give a thickness of 2-7 μm. With proper treatment, the section reveals clear tissue structure and exact antigen location to enable high medical-value pathology researches and retrospective studies. This section type can be stored at 4℃ for long term use.

    3. Processing

      1. Cell on Coverslip
        • Place settled coverslip in culture bottle or perforated plate
        • Take out coverslip after cell growth has reached 60%
        • Fix coverslip with cold acetone or 4% paraformaldehyde for 10 to 30 min
        • Store cell (in gelatin) at -20℃
      2. Cell Smear
        • Collect non-adherent cells and wash 2X by cold PBS buffer
        • Re-suspend cells with PBS buffer
        • Add 30-50 µL to settled slide and smear it evenly
        • Air dry slide a little bit and cover cell with 4% paraformaldehyde for 2-4 hours
        • Store cell (in gelatin) at -20℃
  6. Antigen Retrieval

    Formaldehyde fixation usually generates methylene bridges which cross-link proteins and therefore mask the epitope of interest. It is essential to unmask the antigen epitopes in order to allow the antibodies to bind, either by heat (Heat Induced Epitope Retrieval: HIER) or enzymatic digestion (Proteolytic Induced Epitope Retrieval: PIER).

    1. HIER

      The HIER method can be implemented by microwave, high pressure or water bath. It breaks the methylene bridges and exposes the epitope to allow the antibodies to bind by continuously heating. The following antigen retrieval reagent is required:

      • 0.01 M citrate buffer solution (pH 6.0)
      • 0.01 M PBS buffer (pH7.0)
      • 0.05 M EDTA (pH 8.0)
      • 0.05 M Tris-EDTA (pH 9.0)
      • 0.05 M Tris-HCl (pH 1~12)
      1. Microwave Method
        • Place the sample section into a microwaveable vessel where antigen retrieval reagent is present
        • Place the vessel inside a microwave oven
        • Apply microwave radiation to the sample for 5-20 min
      2. High Pressure Method
        • Place the sample section into an appropriate vessel where antigen retrieval reagent is present
        • Place the vessel inside a pressure cooker
        • Turn on the cooker and heat the sample until it boils
        • Once boiling starts, turn off the cooker after the sample is allowed to reach full pressure for 1-4 min
      3. Water Bath Method
        • Place the sample section into an appropriate vessel where antigen retrieval reagent is present
        • Place the vessel and thermometer inside a water bath chamber
        • Heat the sample to 92℃ in the chamber
        • Remove the sample from the chamber after it is heated at 92℃ for 20-40 min

      Notes

      • The temperature and time should be properly controlled for the antigen retrieval methods described above.
      • To avoid original protein structure restoring, do not cool the sample section by taking it out of the buffer solution.
      • The higher the temperature, the shorter the heating time (vice versa).
    2. PIER

      Epitope can be exposed by incubation with proteases which can break the methylene bridges. The choice for digestion enzymes depends on the antigenic components. Pepsin and bromelin are used for retrieving antigens in intercellular substance. Other enzymes can be used for intracellular antigen exposure.

      Enzyme Working Concentration Digestion Condition
      Trypsin 0.05% to 0.1% 37℃ (10 to 40 min)*
      Proteinase K 20 µg/mL 37℃ (20 min)
      Pepsin 0.40% 37℃ (30 to 180 min)

      * The reaction time can be increased for certain worn-out tissues. Fresh trypsin solution should be prepared with pH adjusted to 7.6 and used at 37℃.

      1. Frozen

        The most important feature for this type of tissue section is to keep antigen’s immune-competence completely, especially for the cell surface antigen. Both fresh and fixed tissues can be processed as frozen tissues. However, the tissues must be dried (or primary fixed) and stored at low temperature.

      2. Paraffin-Embedded

        Paraffin-embedded tissue section is normally sliced by a rotary microtome to give a thickness of 2-7 μm. With proper treatment, the section reveals clear tissue structure and exact antigen location to enable high medical-value pathology researches and retrospective studies. This section type can be stored at 4℃ for long term use.

  7. Detection

    IHC detection methods vary and are based on the nature of analyze reporting and binding chemistry, among other factors. Three methods are described here: immunofluorescence (IF), Enzymatic and Affinity.

    1. Immunofluorescence Method

      Coons and co-workers developed the IF technique in 1941. This technique is used for the rapid identification of an antigen by exposing it to known antibodies labeled with the fluorescent dye (i.e., fluorochrome) which produces light when excited by a laser (e.g. argon-ion laser). Specific antibody binding can be determined by the production of characteristic visible light and detected by a fluorescence microscope. Tables 1 and 2 show some of the common fluorechromes and their corresponding excitation (λex) and emission wavelengths (λem) for nuclear staining and IF, respectively.

      Table 1: Common Fluorochrome for Nuclear Staining

      Fluorochrome λex (nm) λem (nm) Color
      AO 405 530 → 640 Yellowish (Green → Orange)
      DAPI 358 461 Blue
      EB 488 610 Red
      PI 488 620 Red
      Hoechst 33258 352 461 Blue
      Hoechst 33342 352 461 Blue

      Coons and co-workers developed the IF technique in 1941. This technique is used for the rapid identification of an antigen by exposing it to known antibodies labeled with the fluorescent dye (i.e., fluorochrome) which produces light when excited by a laser (e.g. argon-ion laser). Specific antibody binding can be determined by the production of characteristic visible light and detected by a fluorescence microscope. Tables 1 and 2 show some of the common fluorechromes and their corresponding excitation (λex) and emission wavelengths (λem) for nuclear staining and IF, respectively.

      Table 2: Common Fluorochrome for IF Labeling

      Fluorochrome λex (nm) λem (nm) Color
      Alexa 488 488 497 to 643 Green
      Alexa 546 530/545 610/675 Red
      Alexa 647 650 668 Red
      APC  650 660 Red
      B-PE 546, 565 575 Orange, Red
      Cy3 554 570 Red
      FITC 495 525 Green
      RB200 570 596 Orange
      R-PE 480, 546, 565 578 Orange, Red
      Texas Red 596 620 Red
      TRITC 552 570 Red
      1. Principle

        The indirect staining process involves three steps

        • Primary antibody binds specifically to target antigen
        • Secondary antibody labeled with fluorophore binds to primary antibody
        • Fluorophore is detected via microscopy
      2. Protocol
        • Affix the sample on glass slide (To ensure the validity of fluorescence staining, positive, negative and sample autofluorescence controls should be carried out to confirm there is no non-specific binding.)
        • Add properly diluted primary antibody to cover the sample
        • Place the slide into a wet box and incubate at 37℃ for 1-2 hours
        • Wash the slide 3X with 0.01 M PBS (pH 7.4) for 5 min each
        • Remove excess water on the sample (but keep it wet)
        • Cover the sample with properly diluted secondary antibody
        • Place the slide into a wet box and incubate at 37℃ for 30-60 min
        • Wash the slide 3X with 0.01 M PBS (pH 7.4) for 5 min each
        • Remove excess water on the sample (but keep it wet)
        • Add buffered glycerol (mounting medium) to the sample and mount with coverslip
        • View the coverslip under fluorescence microscope
      3. Tips: Operations of Fluorescence Microscope
        • Operate the microscope according to the manual
        • Turn on the mercury lamp for 5-15 min to stabilize the light source before use
        • Wear protective glasses when adjusting light source to avoid harmful ultraviolet rays to eyes
        • Intensity of high pressure mercury lamp will drop if the lamp is used for more than 90 min (Typically, the lamp is continuously used for 1-2 hours)
        • Photo-bleaching occurs if the sample is illuminated by high pressure mercury lamp for more than 3 min (Note: The sample is generally observed within one hour after fluorescence staining)
        • Observe the samples intensively to save time as light source is limited
        • Re-start the light source after turning it off for 30 min or longer
        • Avoid using the light source several times during one day
        • Observe samples immediately after staining
        • There are four levels for fluorescence intensity:
          -: Non or weakly visible autofluorescence
          +: Clearly visible fluorescence
          ++: Brightly visible fluorescence
          +++: Dazzling visible fluorescence
      4. Counterstaining and Stained Sample Storage
        1. Nucleus Counterstaining

          After fluorescence staining, counterstain should be carried out to make morphological structure of cells and tissues well defined and specific fluorescence more easily visible. Some of the counterstaining fluorochromes are:

          • DAPI: classic blue counterstain which is used extensively for nucleus and chromosome staining (DAPI binds selectively to dsDNA without background staining in cytoplasm; DAPI has semi-permeability to living cells and can be used to stain fixed cells and/or tissue sections)
          • Hoechst 33342: primary counterstain which is used against yellow fluorescence
          • Propidium iodide: primary counterstain which is used for nucleus and chromosome staining against yellow/red fluorescence
        2. Stained Sample Storage

          After staining, the samples should be observed and imaged immediately under a fluorescence microscope. They should be mounted in buffered glycerol medium and stored at 4℃ for less than one week if the image is not taken immediately. If anti-fluorescence decay medium is applied to the sample, fluorescence signal may not decay significantly within one month.

    2. Enzymatic Method

      The enzymatic IHC technique was introduced by Nakane and Pierce in 1967. It identifies antigens of interest by exploiting the principle of antibodies binding specifically to antigens. An enzyme label is reacted with a substrate to yield an intensely colored product that can be analyzed. The enzymatic technique was developed with a similar principle to the IF technique but the two are different as an enzyme is used to label the antibody for the enzymatic method. The advantages of enzymatic IHC over IF IHC are:

      • Fluorescence microscope is not required
      • Accurate antigen location is enabled with better contrast ratio
      • Stained samples can be stored for a long time
      • Hematoxylin can be used as counterstain which enhances study of tissue morphology
      • End-product color can be easily identified and observed by light microscope (and also by electron microscopy due to high electron density)
      • Double and multiple stains can be implemented
      1. Labeled-Enzyme Antibody

        For this method, the antibody used for antigen detection has been labeled with the enzyme before the reaction. After reacting with the targeted antigen, the labeled antigen forms an antigen-antibody complex where the enzyme catalyzes a substrate to yield an insoluble colored product. Subsequently, the product can be analyzed by a light microscope or electron microscope. The labeled-enzyme approach can be done by direct or indirect detections.

        1. Direct Detection

          The direct method is a one-step staining method which involves a labeled antibody (e.g. HRP-conjugated antibody) reacting directly with the antigen of interest. The antigen-antibody-HRP complex is then allowed to react with a DAB substrate for staining.

          While the direct method is simple, rapid and highly-specific, it has low sensitivity and a limited range of primary antibodies that are directly labeled. Despite the shortcomings, the direct method is commonly applied to screen monoclonal antibodies before the large-scale manufacturing process.

        2. Indirect Detection

          The indirect method is a two-step process which involves an unlabeled primary antibody (first layer) that binds to the target antigen in the sample and an enzyme-labeled secondary antibody (second layer) that reacts with the primary antibody. The secondary antibody must be raised against the IgG of the animal species in which the primary antibody has been raised. For instance, if the primary antibody is rabbit anti-human IgG, the enzyme labeled secondary antibody could be goat anti-rabbit IgG.

          Comparing to the direct detection, the indirect detection has numerous advantages. First of all, only a relatively small number of standard conjugated (labeled) secondary antibody is needed to be generated for the indirect method. For example, a labeled secondary antibody raised against rabbit IgG, which can be purchased "off the shelf," is useful with any primary antibody raised in rabbit. With the direct method, it would be necessary to label each primary antibody for every antigen of interest. Secondly, the indirect method has greater assay sensitivity. Thirdly, various kinds of controls could be designed and applied with indirect detection.

        3. Unlabeled-Enzyme Antibody
          1. Enzyme Bridge Method

            Described by a publication from T.E. Mason and his colleagues in 1969, this method is based on the binding of an enzyme label to a target antigen through the antigen-antibody reactions of an immunoglobulin-enzyme bridge which consists of the following components in order:

            • Specific antiserum for the tissue antigen (AnTAn)
            • Antiserum against the immune globulin of the species for AnTAn
            • Specific antiserum prepared against the enzyme label in the same species as AnTAn
            • Enzyme label
          2. Peroxidase-Anti-Peroxidase (PAP) Method

            This method involves immunization of a rabbit/goat/rat antibody with a HRP moiety to produce an anti-HRP rabbit/goat/rat antibody which would then bind to another HRP moiety to form a stable polygon. The PAP approach excels due to its high sensitivity and low background for tissue staining.

      2. Protocol
        • Affix the sample on glass slide (To ensure the validity of fluorescence staining, positive, negative and sample autofluorescence controls should be carried out to confirm there is no non-specific binding.)
        • Add properly diluted primary antibody to cover the sample
        • Place the slide into a wet box and incubate at 37℃ for 1-2 hours
        • Wash the slide 3X with 0.01 M PBS (pH 7.4) for 5 min each
        • Remove excess water on the sample (but keep it wet)
        • Cover the sample with properly diluted secondary antibody
        • Place the slide into a wet box and incubate at 37℃ for 30-60 min
        • Wash the slide 3X with 0.01 M PBS (pH 7.4) for 5 min each
        • Remove excess water on the sample (but keep it wet)
        • Add buffered glycerol (mounting medium) to the sample and mount with coverslip
        • View the coverslip under fluorescence microscope
      3. Tips: Operations of Fluorescence Microscope
        • Operate the microscope according to the manual
        • Turn on the mercury lamp for 5-15 min to stabilize the light source before use
        • Wear protective glasses when adjusting light source to avoid harmful ultraviolet rays to eyes
        • Intensity of high pressure mercury lamp will drop if the lamp is used for more than 90 min (Typically, the lamp is continuously used for 1-2 hours)
        • Photo-bleaching occurs if the sample is illuminated by high pressure mercury lamp for more than 3 min (Note: The sample is generally observed within one hour after fluorescence staining)
        • Observe the samples intensively to save time as light source is limited
        • Re-start the light source after turning it off for 30 min or longer
        • Avoid using the light source several times during one day
        • Observe samples immediately after staining
        • There are four levels for fluorescence intensity:
          -: Non or weakly visible autofluorescence
          +: Clearly visible fluorescence
          ++: Brightly visible fluorescence
          +++: Dazzling visible fluorescence
      4. Counterstaining and Stained Sample Storage
        1. Nucleus Counterstaining

          After fluorescence staining, counterstain should be carried out to make morphological structure of cells and tissues well defined and specific fluorescence more easily visible. Some of the counterstaining fluorochromes are:

          • DAPI: classic blue counterstain which is used extensively for nucleus and chromosome staining (DAPI binds selectively to dsDNA without background staining in cytoplasm; DAPI has semi-permeability to living cells and can be used to stain fixed cells and/or tissue sections)
          • Hoechst 33342: primary counterstain which is used against yellow fluorescence
          • Propidium iodide: primary counterstain which is used for nucleus and chromosome staining against yellow/red fluorescence
        2. Stained Sample Storage

          After staining, the samples should be observed and imaged immediately under a fluorescence microscope. They should be mounted in buffered glycerol medium and stored at 4℃ for less than one week if the image is not taken immediately. If anti-fluorescence decay medium is applied to the sample, fluorescence signal may not decay significantly within one month.

    3. Affinity Method

      The IHC sensitivity can be improved by employing a higher number of enzyme molecules bound to the tissue. In this regard, the multiple binding sites between the avidin and biotinylated antibodies have been exploited for IHC signal amplification. Avidin, an egg white protein, has four binding sites for the low-molecular-weight vitamin biotin to form a large lattice-like complex. Beside avidin, there are other methods which involve streptavidin which is a tetrameric biotin-binding protein that is isolated from Streptomyces avidinii. The avidin and streptavidin methods work almost identically as their structures are very similar (they have very little amino acid homology). Avidin-Biotin Peroxidase Complex (ABC) and Labeled Streptavidin Binding (LSB) are the two most widely used affinity methods for amplifying the target antigen signal.

      1. ABC

        The method involves four sequential steps

        • Incubation of primary antibody with tissue sample to allow binding to target antigen
        • Incubation of biotinylated secondary antibody (which has specificity against primary antibody) with tissue sample to allow binding to primary antibody
        • Pre-incubation of biotinylated enzyme (HRP or AP) with free avidin to form large ABC complexes (Biotinylated enzyme and avidin are mixed together in a pre-determined ratio to prevent avidin saturation)
        • Incubation of the above pre-incubated solution to tissue sample
      2. LSB

        This method uses an enzyme-labeled streptavidin to detect the bound biotinylated primary antibody on the tissue section. It can also be applied if the complex in the ABC method is too big for tissue penetration. Due to its smaller size, the enzyme-labeled streptavidin is used to enable tissue penetration. The LSB method can be employed to replace the ABC method for the former’s ability to improve sensitivity and reduce signal further. The information below describes the general staining procedure.

        • Incubation of primary antibody with tissue sample to allow binding to target antigen
        • Incubation of biotinylated secondary antibody (which has specificity against primary antibody) with tissue sample to allow binding to primary antibody
        • Incubation of streptavidin-enzyme conjugate to tissue sample
  8. Chromogens, Counterstains and Mounting Media

    1. Chromogens for HRP

      1. DAB

        DAB (3,3’-Diaminobenzidine) is typically used as a signal enhancer in conjunction with the HRP-based immunostaining systems. The dark brown end-product derived from DAB is insoluble in water and alcohol, stable and suitable for long-term storage. In addition, the end-product could be observed under a light microscope or processed with OsO4 for observation under electron microscopy. Hematoxylin, methyl green and methyl blue are the compatible counterstains. Since DAB may cause skin and bladder cancers, it is advised that personal protective equipment should be used and skin/mucosa should be avoided.

      2. AEC

        After staining with AEC (3-Amino-9-Ethylcarbazole), the positive area on tissue section changes to dark red. The end-product derived from AEC is soluble in organic solvent and cannot be stored on a long-term basis. Similar to DAB, hematoxylin, methyl green and methyl blue are some of the suitable counterstains for AEC. Glycerin gelatin should be used as the AEC mounting medium.

    2. Chromogens for AP

      1. BCIP/NBT

        Used in conjunction, BCIP (5-Bromo-4-Chloro-3-Indolyl-Phosphate)/NBT (Nitro Blue Tetrazolium) is a widely accepted chromogenic substrate used in the AP-based immunostaining systems. After exposing to AP, the substrate changes to bluish violet or black violet. The end-product derived from BCIP/NBT is insoluble in alcohol. Nuclear fast red and brilliant green are the suitable counterstains for BCIP/NBT.

      2. Fast Red TR Salt

        Fast Red is also used for the colorimetric detection of AP. Its end-product has a rose color and is soluble in alcohol. These counterstains are used for the Fast Red chromogens: methyl green, brilliant green and soluble hematoxylin.

    3. Counterstains

      After staining the target antigen by IHC, a secondary stain is usually applied to provide contrast that helps the primary stain more distinct. While many of these stains show specificity for discrete antigens or cellular compartments, other stains will deliver the staining of a whole cell. Some of the most common counterstains are described as follows:

      1. Hematoxylin

        Hematoxylin, a natural dye which is extracted from the heartwood of the logwood tree, is used for cell nucleus staining. Differentiation refers to the process of using reagents (e.g. 1% hydrochloric acid HCl and alcohol) to remove the color caused by overstaining or non-specific staining on sample tissues. After running nucleolar staining (in aluminum hematoxylin) and differentiation (in HCl and alcohol), the tissue section is transferred from an acid solution to an alkaline solution (e.g. ammonia water and disodium hydrogen phosphate solution). During this process, the section will change from red brown into blue. This procedure is known as bluing.

        Hematoxylin is sub-categorized into Mayer’s Hematoxylin and Harris Hematoxylin.

        1. Mayer’s Hematoxylin is reddish violet and is valued for several properties: low staining time, no perception and metal membrane as well as no post-staining differentiation.
        2. Harris Hematoxylin is purple red, widely used in H&E staining and has these advantages: fast staining, bright color, clear nucleolar stains and well defined tissue morphology. Although metallic oxide may float on the Harris hematoxylin solution after a long period of time, filter is unnecessary before use as no perception will appear. Differentiation and bluing should be carried out after staining with Harris Hematoxylin.
      2. Methyl Green

        Methyl green consists of metallic green microcrystals or bright green powders. It becomes bluish green when dissolved in water. This basic dye can be easily bounded with highly polymerized DNA and changes the nucleus to green. Counterstain with methyl green takes 2 to 5 min which should be followed by washing the sample, dehydration and mounting.

      3. Nuclear Fast Red

        This counterstain will change the nucleus to red after applying to the tissue section for 2 to 5 min.

    4. Mounting Media

      A mounting medium may be used to attach a coverslip or may itself be used to replace the coverslip. Generally, the medium selection depends on a few factors including the chemical compatibility with chromogen and counterstain as well as the preservation period.

      1. Neutral Mounting Medium

        It usually refers to an oily substance with pH 7.0 such as neutral gum (resin). Before mounting, the sample should be treated with dimethyl benzene, transparent and dehydrated for long-term storage sections.

      2. Water-Soluble Mounting Medium

        Popularly used in IF staining for short-term storage sections, this mounting medium usually consists of 50% glycerol.

      The table below summarizes the choice of mounting medium among different enzymes, chromogens and counterstains.

      Enzyme Chromogen Counterstain Mounting Medium
      HRP DAB Hematoxylin, Methyl Green, Methyl Blue Neutral
      HRP AEC Hematoxylin, Methyl Blue Water Soluble
      AP BCIP/NBT Nuclear Fast Red, Brilliant Green Neutral
      AP Fast Red Hematoxylin, Methyl Green, Brilliant Green Water Soluble