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.

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

 

  • 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
  • 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
  • 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
  • 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.

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
  • 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.
  • 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
  • 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
  • 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.

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.

  • 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 sa
  • 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