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Immunohistochemistry Handbook

Principle, Troubleshooting, Sample Preparation and Assay Protocols

Introduction


Immunohistochemistry (IHC) combines biochemical, histological and immunological
techniques into a powerful assay for the visualization of protein localization and distribution
within cells in tissue. IHC uses specific antibodies raised against antigens
in cells of a biological sample for detection.


The antibody-antigen binding can be visualized with different methods: enzymes,
such as Horseradish Peroxidase (HRP) or Alkaline Phosphatase (AP), are commonly
used to catalyze a color reaction. There are also numerous IHC methods that
can be used to localize antigens dependent on the specimen types and the assay
sensitivity. Although ICH does not provide quantitative data as with such assays
as Western Blot (WB) or ELISA, IHC can provide a visual context to explain results
obtained in quantitative assays.


A well-known example of demonstrating the power of this method was demonstrated
in a diagnostic cancer screen. The assay was developed on the basis of
research, revealing that a “tumor suppressor” gene associated with cell cycle regulation
is inactivated in many cancers and in turn promotes cell proliferation. IHC and
other experimental techniques have demonstrated that, depending on the type of
cancer involved, this inactivation can occur via degradation, impaired synthesis or
improper localization.


Further, in basic research IHC is used to demonstrate altered localization, processing,
trafficking and targeting of a protein of interest within cells and tissues. For
example, some proteins translocate between different cellular compartments in
response to certain stimuli. Bcl-2, for instance, translocates from the cytoplasm to
the nucleus in response to ischemia following a stroke. The nuclear translocation of
the protein has been convincingly demonstrated by IHC (in combination with other
experiments) before and after cerebral hypoxia when ischemia is induced.

 

IHC experiment design

 

There are a number of important considerations that must be taken into account
and variables that must be optimized in order to consistently obtain robust and
reproducible results from IHC experiments. The selection of suitable antibodies, the
method of tissue fixation, epitope rescue, blocking procedure, antibody concentrations
and suitable controls must all be considered in the design of the experiment.

In the following guide, we address these points and suggest sample protocols to
help with the design of IHC experiments.

Choose the right antibody

 

Variable

 

 

 

Options

 

 

Considerations

 

 

 

 

 

 

 

 

Antibody Type

 

 

Monoclonal

 

 

Advantages

• Mouse or rabbit hybridoma

• Good consistency

 

Disadvantages

• Lower affinity

• False negatives common

 

 

 

 

 

 

 

Polyclonal

 

 

Advantages

• Several different species

 

Disadvantages

• More non-specific reactivity

• Affinity may vary between lots

 

 

 

 

 

Antibody Target

 

 

Entire Molecule

 

• Depending on location of target antigen

e.g., If C-terminus is embedded in a membrane, it is not a good choice for a target.

 

 

 

 

 

C-Terminus

 

 

 

N-Terminus

 

 

Table 1. Factors involved in selecting suitable IHC antibodies.

 

Monoclonal vs. Polyclonal antibodies

Monoclonal antibodies are generated from a single B-cell clone from one animal,
which produces immunoglobulins specific against a single epitope. These clones
are fused to a myeloma cell (B-cell cancer) to create an immortal cell called a hybridoma,
which produces large amounts of homogenous antibodies.

Monoclonal antibodies are easy to standardize as they show reliable consistency between batches,
however the targeting of a single epitope means less cross-reactivity with other
proteins which can cause false negative results.

Polyclonal antibodies are created using multiple B-cell clones of one animal, which
results in a heterogeneous mix of antibodies that target several epitopes of the
same antigen.

As a result, polyclonal antibodies are more forgiving of changes in
target proteins that can easily result from the fixation process. The fact that they
recognize multiple epitopes also means that they can amplify the signal in samples
in which the protein of interest is expressed at low levels. This feature also makes
polyclonal antibodies the antibodies of choice when dealing with denatured proteins.

Further, while monoclonal antibodies are generated from mouse and rabbit hybridomas,
polyclonal antibodies are raised in several different species including rats,
goats, sheep, guinea pigs, chickens and more.


It is important to bear in mind that the primary antibody should be raised in a host
specie as different from the source of your samples as possible. Otherwise, IgG
secondary antibodies will bind to all the endogenous IgG in the tissue, causing high
background signal.

 

Region of Targeted Protein

Antibodies are generated by using an array of immunogenic substances to induce
an immune response in the host animal. These immunogenic substances may include
full-length proteins, protein fragments, peptides and whole organisms such
as bacteria or cells.

If the target epitope is a protein fragment or a specific isoform
or region of a full-length protein, the antibody used must be raised against an
epitope that is identical to or contained within the fragment or region. For example,
if the target is a membrane protein, the appropriate antibody should target the protein’s
extracellular domain.


Also some protein families contain a conserved motif consisting of a sequence of
amino acids, where small variations within this motif can be exploited to differentiate
between subtypes of proteins within the family.

 

Principle

 

In order to be able to use certain antibodies, samples must be processed or fixed
in a very specific manner. Many antibodies only recognize proteins that have been
reduced and denatured, while others only detect protein epitopes when the proteins
are in their native, folded conformation.

Some antibodies can only be used with unfixed, frozen tissue as they cannot bind to their target epitopes in formalinfixed, paraffin-embedded tissues. However, an antigen retrieval step can be added
to reverse the cross-links that result from fixation. If your protein of interest is located intracellularly, the cells must be permeabilized with solvents or detergents prior to enable the antibodies access to the interior of the cell.

Largely, detergents are the most popular method for permeabilizing cells,
however, because they disrupt the membrane, detergents are not appropriate for
cytoskeletal antigens or low molecular-weight targets such as viral antigens and certain
enzymes.

ihc.JPG

[Figure 1. IHC Workflow from Sample to Results.]

In this handbook, the following topics will be covered:
1. Fixation
2. Tissue Sectioning
3. Paraffin Embedding
4. Inactivation and Blocking
5. Antigen Retrieval
6. Detection
7. Chromogens, Counterstains and Mounting Media

 

Fixation

Fixation preserves morphology, prevents tissue degradation, ensures antigenicity and helps to protect cells from damage during the tissue preparation process. The reproducibility and quality of IHC results are highly dependent on fixation times, temperature, pH and buffer composition—all of which must be carefully optimized and standardized for different starting materials.
Selection of Fixing Solution
In the following commonly used fixing solutions are listed, which need to be tested whether a specific type of solution is appropriate for your detected antigen, as there is no standard fixing solution for different kinds of antigen immobilization.

(i) Acetone and Alcohol
Acetone and alcohol are both primary fixing solutions, which play a role in precipitating sugars and fats, as well as maintaining the immunologic competence. Note that alcohol is ineffective in maintaining low molecular weight protein, polypeptide and cytoplasmic proteins. However, alcohol can be mixed with glacial acetic acid, ethyl ether, chloroform and formaldehyde to overcome this downside. Acetone on the other hand is often used for frozen tissue and cytological smears as it has a strong
penetrability and dehydration property.
(ii) Aldehyde
Aldehydes are di-functional cross-linking agents, which are widely used due to their strong penetrability, low contractibility and low background. They provide the cross-linking between tissues and maintain the antigen. While formalin (saturated 37% formaldehyde solution) is the most widely used, 4% paraformaldehyde is better than formaldehyde as it’s the pure form of formaldehyde and methanol-free. Further, Bouin’s solution (containing picric acid) is the most widely used in histology and pathology, whereas Zamboni’s solution is applied to light and electron microscopic immunocytochemistry and is more suitable than formaldehyde in maintaining ultra structural organization.
(iii) Non-Aldehyde
Carbodiimide, dimethylacetamide, dimethyl-suberimidate and para-benzoquinone are widely used for the fixation of peptide hormones in tissues and are better mixed with glutaric dialdehyde or paraformaldehyde.

Fixation Methods
(i) Fixation by Perfusion
As fixation can cause cross-linking, which masks epitopes, the best way to preserve tissue morphology and target protein antigenicity is to replace the animal’s entire systemic blood volume by fixative with vascular perfusion. This is followed by perfusion with sucrose solution.
(ii) Fixation by Immersion
For the majority of applications, it is not to feasible to perform fixation through perfusion, however, an alternative method is to immerse tissues in fixative solution. Biopsy and surgical specimens, as well as other non-irrigation tissues commonly employ this fixation method. The fixation duration is generally determined by the antigen stability and the type of fixing solution used.
(iii) Snap Freezing
For cases in which standard fixation techniques cannot be used or fixation will be carried out later, tissue can be snap frozen and kept at -70°Cuntil use. For example, phosphorylation-dependent epitopes have been shown to translocate from the membrane to the cytoplasm following fixation with formaldehyde.
Exercise caution when fixating tissues
- Do not over-fix the tissues
- Store tissues appropriately after fixation
- Use adequate amounts of fixation solution and wash sufficiently after fixation
- Reduce the tissue sample size to less than 2cm×1.5cm×0.3cm (Thickness <0.3 cm), if possible.

Tissue Sectioning

(A) Slide Pre-Treatment
The following pre-treatment is designed to prevent dislocation of the sample from object mount due to elevated temperature, high pressure, radiation or other factors.
(i) Microscopic Slide
In order to remove any oil residues attached to the surface of a glass slide, it should be immersed in cleaning solution for 12 to 24 hours. After washing the slide thoroughly with distilled water, dip it into 95% alcohol for 2 hours and dry by wiping with lens paper or in an infrared oven. Avoid scratching the slide during the whole procedure.
(ii) Cell Samples
(a) Adherent Cells
Cell Climbing: adherent cells are grown on multi-aperture culture plates with coverslips*, culture vessels or chamber slides. The coverslip pre-treatment procedure should be carried out as for microcopy slides except that dipping and cleaning can be completed in 2 hours due to the reduced thickness.Direct Cell Culture: culture adherent cells directly on culture vessels or multi-aperture
culture plates

(b) Non-Adherent Cells
Cell Smear: use chemical bond to adhere the cells on a
Eccentric Cell Smears: adhere the non-adherent cells to culture vessels by cell microcentrifugation
(iii) Tissue Section Mounting
For this application, coat the microscopic slide with 3-Amino Propyl Tri-ethoxy Silane
(APES) in acetone and adhere the tissue by adding a drop of mounting medium
(Glycerol, Gelatin). Further, incubate the coverslip at 45°, allowing the drop to spread
along the edge of the slip and slowly cover the tissue entirely with the coverslip If
necessary, incubate the slide at 80°C for 1 hour.


(B) Tissue Section Types
Tissue samples are typically taken from specimens of various sources: biopsy, surgery,
animal model and autopsy. Autopsy samples are taken after an animal has died
and postmortem autolysis has set in. As antigens may denature, disappear and diffuse,
autopsy specimen especially should be fixated as soon as possible to avoid
degradation of the antigen.
(i) Frozen
The most important feature for this type of tissue section is to keep the antigen’s
immune-competence intact, especially for cell surface antigens. Both, fresh and fixed
tissues can be processed frozen. However, the tissues must be dried (or primary
fixed) and stored at low temperature.
(ii) Paraffin-embedded
Paraffin-embedded tissue is usually sliced at a thickness of 2-7 μm using a microtome.
With appropriate staining, the section reveals clear tissue structure and
exact antigen location for high-resolution microscopy. This type of section can be
stored at 4°C for long-term use.

Take note when collecting, fixating and sectioning the samples:
- Use a sharp or scissors to avoid extrusion damage
- The microtome cutter should be flat, small and thin (Normal size is 1.0 cm ×
1.0 cm × 0.2 cm)
- Eliminate fat tissue and calcifications
- Include normal tissue as control if necessary
- Prepare paraffin-embedded tissue or frozen tissue immediately after sectioning
or store the tissues in liquid nitrogen or at -70°C

Paraffin Embedding

The process of paraffin embedding involves fixation, dehydration, transparentizing, immersion and embedding.
(A)Fixation
Please refer to the fixation section described above (3.1 Fixation).


(B) Dehydration
The dehydration process generally removes water and hardens the tissue. The dehydrating
agents describes below are water-soluble and can be prepared in different
volumetric ratios.
(i) Ethanol
Ethanol is the most commonly used dehydrating agent, achieving strong water
separation and tissue hardening. However, since ethanol has strong penetration and
contractility, its concentration should be progressively increased to avoid excessive
shrinking of the tissue.
(ii) Acetone
As a usual substitute for alcohol, acetone acts as both a fixation and dehydration
agent. Note that the dehydration time with acetone has to be adjusted for each tissue
type, as it tends to over harden tissues.


(C) 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 in the following steps. The addition of transparent reagent helps the paraffin absorb into the tissue. Common transparent reagents are:(i) 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 period of time or it will become brittle and too hard.
(ii) 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 show high toxicity and must be handled with care.
(iii) 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 achieve complete penetration.
(iv) Cedar Oil
Due to minimal clarification created by cedar oil, it is an appropriate transparent agent for fine and soft tissues. Moreover, particularly hard, dense and fibrous tissues are easier to section after immersing in cedar oil. However, this oil is not useful for other common tissue sections due to its high concentration and weak penetrability.


(D) Immersion
After transparentizing, the tissue can be immersed in molten paraffin wax allowing the absorption of the wax-substituting transparent agent. Based upon the melting point of wax, immersion should be performed at 54-64°C.


(E) Embedding
During the process of embedding, the paraffin-infiltrated samples from the previous step are individually enclosed in mesh cassettes, with a specific orientation in the middle of the block for subsequent sectioning. Following, the cassette is filled with molten paraffin and left to cool down. After cooling is completed, the tissue will be ready for sectioning and suitable for storage.

The treatment conditions (using ethanol and xylene as an example) are shown in the
table below.

Step

Reagent

Time

1

75% Ethanol

30 min to 2 h

2

85% Ethanol

30 min to 2 h

3

95% Ethanol

2 h

4

95% Ethanol

2 h

5

95% Ethanol

2 h

6

100% Ethanol

30 mins to 1 h

7

100% Ethanol

30 mins to 1 h

8

100% Ethanol

30 mins to 1 h

9

Xylene

15 min

10

Xylene

15 min

11

Xylene

15 min

12

Paraffin Wax

30 min

13

Paraffin Wax

1 h to 2 h

14

Paraffin Wax

1 h to 2 h

Table 2. Paraffin Embedding Treatment Conditions

(A) Inactivation
The activity of horseradish peroxidase (HRP) or alkaline-phosphatase (AP) used for signal visualization requires blocking or inhibition of endogenous enzymes to avoid non-specific binding and hence artifactual signal.
(i) 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
- Incubate the sample section in 0.1mM Levamisole (Note: Levamisole cannot inhibit the AP activation of endogenous enzymes in testine tissue)
- Incubate the frozen section or cell section in solution composed of methanol
and 3% H2O2 (v/v:4:1) for 30 min

(ii) Endogenous AP Inactivation
- Incubate the sample section in 0.1mM Levamisole
(Note: Levamisole cannot inhibit the AP activation of endogenous enzymes in testine
tissue)


(B) 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 those sites. The blocking should be carried out at room temperature for 10-30 min (avoid excessive blocking).

Antigen Retrieval

Formaldehyde fixation usually generates methylene bridges which cross-link proteins and therefore mask the epitope of interest. It is essential to recover the antibody 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). To find the optimal antigen recovery method, we suggest to test both HIER and PIER methods, compare their results and optimize the method as needed.


(A) HIER
The HIER method can be implemented by microwave, high pressure or water bath.
It breaks the methylene bridges and exposes the epitopes to allow the antibodies
to bind by continuous heating. Comparing to PIER, HIER has a gentler experimental
condition in which users have more control over the experimental parameters. However,
the pH and buffers for HIER must be optimized.
The following antigen retrieval reagents are required:
- M citrate buffer solution (pH 6.0)
- 0.01 MPBS buffer (pH7.0)
- 0.05 M EDTA (pH 8.0)
- 0.05 M Tris-EDTA (pH 9.0)
- 0.05 M Tris-HCl (pH1~12)

(i) Microwave Method
- Place the sample section in a microwaveable vessel with antigen retrieval reagent
- Place the vessel inside a microwave oven and heat up the sample for 5-20 min
(ii) High Pressure Method
- Place the sample section in an appropriate vessel with antigen retrieval
- Place the vessel inside a pressure cooker and heat the sample until it boils
- Once boiling starts, turn off the cooker after the sample is allowed to reach fullpressure for 1-4 min
(iii) Water Bath Method
- Place the sample section in an appropriate vessel with antigen retrieval reagent
- Place the vessel and thermometer inside a water bath chamber and heat the sample to 92°C in the chamber
- Remove the sample from the chamber after it is heated at 92°C 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 (and vice versa).


(B) PIER
Epitopes can be exposed by incubation with proteases, which enzymatically digest the methylene bridges. The choice for digestion enzymes depends on the antigenic components. Pepsin and bromelin are used for retrieving antigens in intercellular. Epitopes can be exposed by incubation with proteases, which enzymatically digest 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. PIER is suitable for retrieving more difficult epitopes, while the pH for incubation is usually known. However, PIER is a harsher method and can damage tissue morphology.

Enzyme

Working Concentration

Digestion Concentration

 

Trypsin

 

 

0.05% to 0.1%

 

37°C (10 to 40 min)*

Proteinase

 

20 μg/mL

 

37°C (20 min)

 

Pepsin

 

0.4%

 

37°C (30 to 180 min)

 

Table 3. Digestion Enzymes for PIER.
* 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°C.

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.
(A) Immunofluorescence Method

 

 

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

 

Table 4: Common Fluorochromes for Nuclear Staining. Fluorochrome

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 fluorochromes and their corresponding excitation (λex) and emission wavelengths (λem) for nuclear
staining and IF, respectively.

 

λex (nm)

λem (nm)

 

Colour

 

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

Table 5: Common Fluorochromes for IF Labeling.

(B) Fluorochrome
(i) Principle
The indirect staining process involves three steps
- Primary antibody binds specifically to a target antigen
- Secondary antibody labeled with fluorophore binds to primary antibody
- Fluorophore is detected via microscopy
(ii) 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
- Intensity of high pressure mercury lamp will drop if the lamp is used for more than 90 min (typically, the lamp is continuously used for1-2 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 the light source is limited
- Re-start the light source after turning it off for 30 min or longer
(iii) Counterstaining and Stained Sample Storage

(a) Nuclear Counterstaining
After the fluorescence staining, a counterstain should be carried out to visualize the overall structure of cells and tissues.
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
(b) Stained Sample Storage
Following the staining, the samples should be observed and imaged immediately under
a fluorescence microscope. If the imaging cannot be carried out immediately, the
samples can be mounted in buffered glycerol medium and stored at 4°C for less one
week. If anti-fading medium is applied to the sample, fluorescence signal may not decay
significantly within one month.

(B) Enzymatic Method
The antigen of interest is detected with a specific antibody, which is labeled with an enzyme. The enzyme label later reacts with a substrate to yield a colored product. The enzymatic technique was developed with a similar principle to the IF technique, but they differ as an enzyme is used to label the antibody for the enzymatic method. The advantages of enzymatic IHC over IF IHC are:
- A fluorescence microscope is not required
- Accurate antigen location is enabled with better contrast ratio
- Stained samples can be stored for a long time
- Haematoxylin can be used as a counterstain, which enhances study of tissue morphology
- The colorimetric signal can be easily identified and observed by light microscopy
- Double or multiple stains can be implemented

(i) 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 with
microscopy or electron microscopy. The labeled-enzyme approach can be done by
direct or indirect detections.

[Figure 4. Direct vs. Indirect IHC Detection Methods.]

(a) 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 able 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.
(b) Indirect Detection
The indirect method is a two-step process, which involves an unlabeled primary antibody that binds to the target antigen in the sample and an enzyme-labeled secondary antibody 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 secondary antibodies are needed 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. Moreover, various kinds of controls could be designed and applied with indirect detection.

(ii) Unlabeled-Enzyme Antibody

(a) Enzyme Bridge Method
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
(b) Peroxidase-Anti-Peroxidase (PAP)Method
This method involves immunization of a rabbit/goat/rat antibody with a HRP component to produce an anti-HRP rabbit/goat/rat antibody, which would then bind to another HRP part to form a stable polygon. The PAP approach excels due to its high sensitivity and low background for tissue staining.

(C) 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 that employ 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. Avidin-Biotin Peroxidase Complex (ABC) and Labeled Streptavidin Binding (LSB) are the two most widely used affinity methods for amplifying the target antigen signal.

(i) ABC

[Figure 5. ABC Detection Method.]

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

(ii) LSB

[Figure 6. LSB Detection Method.]

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 enzymelabeled 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


3.7 Chromogens, Counterstains and Mounting Media


(A) Chromogens for HRP

(i) DAB
DAB (3,3’-Diaminobenzidine) is typically used as a signal enhancer in conjunction with the HRP-based immunostaining systems. The dark brown product derives from DAB is insoluble in water and alcohol, stable and suitable for long-term storage. In addition, the staining product could be observed with light microscopy or processed with OsO4 for observation with electron microscopy. Haematoxylin, methyl green and methyl blue are the suitable counterstains. Since DAB may cause skin and bladder
cancers, it is necessary to use personal protective equipment to avoid contact with skin or mucosa.
(ii) AEC
After staining with AEC ( 3-Amino-9-Ethylcarbazole), the positive area on tissue section changes to dark red. The product derived from AEC is soluble in organic solvent and cannot be stored on a long-term basis. Similar to DAB, haematoxylin, methyl green and methyl blue are some of the suitable counterstains for AEC. Glycerin gelatin should be used as the AEC mounting medium.

(B) Chromogens for AP
(i) 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 a blue/ violet stain. The staining product derived from BCIP/NBT is insoluble in alcohol. Nuclear fast red and brilliant green are the suitable counterstains for BCIP/NBT.
(ii) Fast Red TR Salt
Fast Red is also used for the colorimetric detection of AP. Its product has a rose color and is soluble in alcohol. Methyl green, brilliant green and soluble haematoxylin are used for the Fast Red chromogen counterstains.

(C) Counterstains
After staining the target antigen by IHC, a secondary stain is usually applied to provide information about the overall morphology of the sample. 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:
(i) Haematoxylin
Haematoxylin, a natural dye, 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 overstraining or non-specific staining on sample tissues. After running nuclear staining (in aluminum haematoxylin) 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, which is known as bluing. Haematoxylin is sub-categorized into Mayer’s Haematoxylin and Harris Haematoxylin.
(a) Mayer’s Haematoxylin is reddish violet and is valued for several properties: low
staining time, no perception and metal membrane as well as no post-staining differentiation.
(b) Harris Haematoxylin is purple red, widely used in H&E staining and has these advantages:
fast staining, bright color, clear nuclear stains and well defined tissue morphology. Although metallic oxide may float on the Harris haematoxylin solution after a long period of time, filtering is unnecessary before use as no precipitation will appear. Differentiation and bluing should be carried out after staining with Harris Haematoxylin.

(ii) Methyl Green
Methyl green consists of metallic green microcrystalsor 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.

(iii) Nuclear Fast Red
This counterstain will change the nucleus to red after applying to the tissue section for 2 to 5 min.

(D) Mounting Media
A mounting medium may be used to attach a coverslip or may be used to replace the coverslip. Generally, the medium selection depends on a few factors including the chemical compatibility with chromogens and counterstains, as well as the preservation period.
(i) 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 dimethylbenzene, transparent and dehydrated for long-term storage sections.
(ii) Water-Soluble Mounting Medium
Popularly used in IF staining for short-term storage sections is 50% glycerol in water. The table below summarizes the choice of mounting medium among different enzymes, chromogens and counterstains.

 

Enzyme

Chromogen

Counterstain

Mounting Medium

HRP

DAB

Haematoxylin, Methyl Green,

Methyl Blue

Neutral

HRP

AEC

Haematoxylin, Methyl Blue

Water Soluble

AP

BCIP/NBT

Nuclear Fast Red, Brilliant Green

Neutral

AP

Fast Red

Haematoxylin, Methyl Green, Brilliant Green

Water Soluble

Table 6. Chromogens, Counterstains and Mounting Media for HRP and AP Enzymatic Systems.

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