Antibody-mediated target antigen detection, or staining, is the keystone of immunohistochemistry, and this article discusses the factors that are critical to obtaining optimum results.

Antibody diluents and rinse buffers

For IHC staining, antibodies should be diluted with buffer solutions that stabilize the antibodies during staining and long-term storage. These buffers often contain bovine serum albumin (BSA; 0.2–5%) or some other protein-based stabilizer/carrier protein dissolved in phosphate buffered saline (PBS) at pH 7–7.5. A small amount of detergent, such as Tween 20, is also usually added at 0.01–0.1% (v/v) to assist in uniform wetting of the sample.

Thorough sample washing between antibody treatments is critical to remove unbound and weakly bound antibody. Common wash or rinse buffers consist of a small amount of a gentle surfactant, such as Tween 20 (0.01–0.2%), diluted in PBS, Tris-buffered saline (TBS) or even just distilled water. Multiple washing steps are recommended in between each staining step.


Reporter selection

Enzyme reporters and chromogenic substrates

Antibody-mediated antigen detection requires a reporter to visually identify the target antigen, and the type of reporter used is determined by many factors, including:

  • the type of experiment
  • the level of antigen expression
  • whether or not signal quantitation is required
  • the kinds of imaging needed (light microscope vs. epifluorescence vs. confocal microscope)
  • the cost of reagents and equipment

The most popular methods of detection are with enzyme- and fluorophore-mediated chromogenic and fluorescent detection, respectively. With chromogenic reporters, an enzyme label is reacted with a substrate to yield an intensely colored product that precipitates at sites where antigen is found. Chromogenic results can be imaged with an ordinary light microscope. Alkaline phosphatase (AP) and horseradish peroxidase (HRP) are the two enzymes used most extensively as labels for protein detection. An array of chromogenic, fluorogenic and chemiluminescent substrates is available for use with either enzyme.

Horseradish peroxidase (HRP) is a 44-kDa protein that catalyzes the oxidation of substrates in the presence of hydrogen peroxide, resulting in a colored product or the release of light as one product of the reaction (chemiluminescence). HRP functions optimally at a near-neutral pH and can be inhibited by cyanides, sulfides and azides. Antibody–HRP conjugates are superior to antibody–AP conjugates with respect to the specific activities of both the enzyme and antibody. In addition, its high turnover rate, good stability, low cost and wide availability of substrates makes HRP the enzyme of choice for most IHC applications.

Alkaline phosphatase (AP), usually isolated from calf intestine, is a 140-kDa enzyme that catalyzes the hydrolysis of phosphate groups from a substrate molecule, resulting in a colored product or the release of light as one product of the reaction (chemiluminescence). AP has optimal enzymatic activity at a basic pH (pH 8–10) and can be inhibited by cyanides, arsenate, inorganic phosphate and divalent cation chelators, such as EDTA. In the example shown below, human colon carcinoma cells were probed for cytokeratin 18 protein using chromogenic IHC techniques. 

Chromogenic methods for target antigen detection. Human colon carcinoma sections were stained for cytokeratin 18 through indirect detection using Invitrogen biotin-conjugated goat anti-rabbit secondary antibody (Cat. # A16108), poly-HRP conjugate (Cat. # 32260) and Thermo Scientific Metal Enhanced DAB substrate (Cat. # 34065) to develop the signal.


Characteristics of common chromogenic reporters used for IHC
Enzyme label Substrate Reporter color
Horseradish peroxidase (HRP) 3,3'-diaminobenzidine (DAB) Brown to black
Horseradish peroxidase (HRP) Aminoethyl carbazole (AEC)  Red
Alkaline phosphatase (AP) Fast Red Red 
Alkaline phosphatase (AP) Combination of nitro blue tetrazolium chloride (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) Black to purple
Glucose oxidase (GO)*  Nitro blue tetrazolium chloride (NBT) Blue to purple
β-galactosidase (BGAL)* 5-bromo-4-chloro-3-indoyl-β-D-galactopyranoside (BCIG or X-Gal)  Blue

*These enzymes are rarely used nowadays for IHC and few GO and BGAL conjugates are commercially available.

Fluorescent reporters

Organic fluorophores or fluorescent dyes are conjugated to the primary or secondary antibody and are frequently used in IHC. As mentioned previously, fluorophore-based detection with tissue sections is really immunofluorescence (IF) detection. It is not IHC per se, even though we and others use IF and IHC as interchangeable terms. Fluorescent reporters do not require a substrate to activate an enzyme, and high-resolution fluorescence microscopy can be performed to detect the antigen. An added benefit of fluorescent reporters is that individual fluorescent colors can be assigned to individual antigens for multiplex staining.

Use of fluorescent IHC for target antigen detection. Human colon carcinoma sections were stained for cytokeratin 18 through direct staining with biotinylated primary antibody (Cat. # MA5-12104) and followed by incubation with Invitrogen DyLight 594–conjugated streptavidin (Cat. # 21842) for fluorescent detection.


Direct vs. indirect staining

Antibody-mediated antigen detection approaches are separated into direct and indirect detection, and the choice of which approach to use is dependent upon the level of target antigen expression.

Schematic of direct and indirect detection methods.


Direct antibody detection

In direct antibody detection, the antibody against the target antigen (primary antibody) is conjugated to an enzyme, most often HRP or AP, which is then activated by adding appropriate substrates. Alternatively, the primary antibody is conjugated to a fluorophore for detection by fluorescence microscopy. While direct detection does not require the additional step of adding a secondary antibody, there is little signal amplification possible. Therefore, the signal may be difficult to detect if the target antigen is present at low levels in the sample. Also, the conjugation process may in some cases interfere with the ability of the primary antibody to react with the target antigen. A major advantage of this method, though, is that multicolor fluorescence staining is only limited by the number of different fluorophores that the detection system (fluorescence microscope) can detect and not by the availability of the different primary antibodies raised in various species, which can often be restricted to only one.

Indirect antibody detection

The indirect method of detection employs a secondary antibody, which reacts with the unlabeled primary antibody to amplify the detection signal. This amplification is possible because multiple secondary antibodies can bind to a single primary antibody, and thus the sensitivity of detection of target antigens increases with the addition of secondary antibodies. The most popular methods of detection are with enzyme- and fluorophore-conjugated secondary antibodies. After multiple secondary antibodies bind to each primary antibody, the enzyme label (usually HRP or AP) is then reacted with substrate to yield the chromogenic response. A further advantage of using enzyme-labeled systems is the option to make the product electron-dense for electron microscopy. The following image provides an example of indirect and direct IHC staining methods. 

Indirect IHC staining amplifies the target signal over direct methods. Human colon carcinoma tissue samples were fixed and stained for cytokeratin 18 either by direct detection using a biotin-conjugated anti–cytokeratin 18 antibody (Cat. # MA5-12101, right panel) or by indirect detection using a primary anti–cytokeratin 18 antibody (Cat. # MA5-12104) and Invitrogen biotin-conjugated goat anti-mouse antibody (Cat. # 62-6540, left panel). In both approaches, Pierce DyLight 650–conjugated streptavidin (Cat. # 84547B) was used to fluorescently detect the target antigen.


Indirect methods of antigen detection

Avidin is a tetrameric protein naturally found in egg white that binds with high affinity and specificity to biotin. The Avidin-Biotin Complex (ABC) method of antigen detection exploits this binding affinity by using a secondary antibody conjugated to biotin to amplify the signal from the primary antibody. HRP or AP is conjugated to a large avidin–biotin complex, which is then added to the sample to enzymatically label each complex and further amplify the signal. Nowadays, most researchers use conjugates made with streptavidin instead of avidin because the former yields less nonspecific binding.

The Labeled Streptavidin Biotin (LSAB) method builds upon the ABC method by conjugating the enzyme reporter directly to streptavidin. The complexes that are formed are smaller than the large complexes that are formed via the ABC method, and therefore difficult-to-reach epitopes are more easily tagged with LSAB. Evidence shows that LSAB can increase the sensitivity of detection up to ~8-fold over the traditional ABC method.

The Phosphatase-Anti-Phosphatase or Peroxidase/Anti-Peroxidase (PAP) method offers greater sensitivity because the approach uses an additional level of amplification over ABC and LSAB. In PAP, an unconjugated secondary antibody is added to the primary antibody, resulting in multiple secondary antibodies bound to each primary antibody. A tertiary antibody complexed with peroxidase is then added, and because multiple tertiary antibodies will bind to each secondary antibody, the level of amplification upon substrate activation can be as much as 100 to 1,000 times greater than secondary antibody–mediated amplification. Although it requires an additional antibody (anti-phosphatase or anti-peroxidase), an added benefit of PAP is that less primary antibody can be used for each sample staining. The ease of use and availability of multiple types of LSAB reagents have largely supplanted the PAP-based methods.

Schematic of the indirect detection methods for IHC.


Substrates

Immunoenzymatic tissue staining is the result of the reaction of a soluble substrate with an enzyme, which produces an insoluble, colored product that is deposited at antigenic sites. The intensity of the color produced when the substrate is added should correlate with the concentrations of the primary antibody and the respective tissue antigen it binds to. Many enzymes have been used for these applications, but the most common selections are HRP and calf intestinal alkaline phosphatase. Enzymatic activity is dependent on several variables, including enzyme and substrate concentration, buffer, pH, temperature, and possibly light levels.

Substrates range from basic formulations that have been the standard for years to kits that have been optimized for high sensitivity and low background signal.


Optimization strategies

Signal amplification can be further enhanced by Tyramide Signal Amplification (TSA), which may be used in conjunction with HRP-conjugated detection systems. This approach utilizes the catalytic activity of the HRP conjugate to increase the amount of dye or label localized to the antigen-bound primary antibody. Briefly, after the primary and HRP-conjugated secondary antibodies are complexed to the antigen, tyramide derivatives conjugated to a protein probe are added to the sample. The tyramide derivatives react with the peroxidase conjugated to the secondary antibodies to form highly reactive tyramide radicals, which covalently bind to any tyrosine residues in proximity to the short-lived activated tyramide. Once bound, detection of the tyramide-conjugated protein probe is performed using peroxidase-conjugated antibodies specific to the probe, or peroxidase-conjugated streptavidin if the probe is biotin. TSA systems are reported to increase the detection of rare proteins by up to ~100-fold.    The disadvantages of this approach are the high cost of the proprietary TSA kits, the high level of optimization that is often required, and a possible increase in background staining.

Avidin (AV) is positively charged and glycosylated, which can result in high background staining due to both nonspecific binding and interaction with sugar-binding lectins in the tissue sample. Streptavidin (SA), while evolutionarily unrelated to avidin, shows a similar high affinity for biotin. It can reduce high background staining because it is not glycosylated and is only slightly negatively charged. Pierce NeutrAvidin (NA) protein is a form of avidin that is deglycosylated and modified to have a neutral charge. These modifications can further reduce the nonspecific binding, which can result in higher than acceptable background staining.

Biotin-binding conjugates are normally limited to carrying 1 to 3 HRP molecules per protein in order to maintain enzymatic activity. Because of the small size of HRP (44 kDa), further increases in sensitivity may be achieved by using polymeric HRP (poly-HRP)–conjugated secondary antibodies that may eliminate the need for using ABC-type amplification systems for some researchers. Poly-HRP increases the sensitivity to detect proteins present in very low amounts (pg to fg) by increasing the amount of active HRP on the conjugate. This innovation is achieved through proprietary techniques that maintain high enzymatic activity.

Recommended reading
  1. Coons AA et al. (1942) J Immunol  45:159–170.
  2. Beisker W et al. (1987) Cytometry  8:235–239.
  3. Cowen T et al. (1985) Histochemistry  82:205–208.
  4. Mosiman VL et al. (1997) Cytometry  30:151–156.
  5. Romijn, Herms J et al. (1999) J Histochem Cytochem  47:229–236.