Antibody-mediated target antigen detection is the keystone of immunohistochemistry, and the following are critical factors in obtaining optimum results.

Antibody Diluents and Rinse Buffers

For IHC staining, antibodies are diluted into buffer solutions that stabilize the antibodies during staining and long-term storage. These solutions are often made with bovine serum albumin (BSA; 0.2 to 5%) or some other proteinaceous stabilizer dissolved in phosphate buffered saline (PBS). A small amount of detergent (0.1%), such as Tween 20, is also added to assist in uniform spreading across the sample.

Thorough sample washing between antibody treatments is critical not only to remove unbound antibody, but also to wash away antibodies that are weakly bound to nonspecific sites. Common rinse buffers consist of a small amount of detergent (0.2%), such as Tween 20, diluted into PBS, Tris buffered saline (TBS) or even just distilled water. Multiple washings 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 vs. epifluorescent vs. confocal)
  • 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 can be analyzed 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.

Alkaline phosphatase, usually isolated from calf intestine, is a 140-kDa protein that catalyzes the hydrolysis of phosphate groups from a substrate molecule resulting in a colored product or the release of light as a byproduct of the reaction. 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.

Horseradish peroxidase is a 40-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 a byproduct of the reaction. 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 applications.

Characteristics of common chromogenic reporters used for IHC

Enzyme Label Substrate Reporter color
Horseradish peroxidase (HRP) 3,3'-diaminobenzidine (DAB) Brown to black
Alkaline phosphatase (AP) Combination of nitro blue tetrazolium chloride (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) Black to purple
Glucose oxidase Nitro blue tetrazolium chloride (NBT) Blue to purple
β-galactosidase 5-bromo-4-chloro-3-indoyl-β-D-galactopyranoside (BCIG or X-Gal) Blue

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Fluorescent Reporters

Organic fluorophores are conjugated to a probe or the primary or secondary antibody and are increasingly used in IHC. These reporters do not require a substrate to activate an enzyme, and high-resolution fluorescent microscopy can be performed to detect the antigen. An added benefit of fluorescent reporters is that individual colors can be assigned to individual antigens for multiplex staining.

Chromogenic and fluorescent IHC methods for target antigen detection. Human colon carcinoma sections were stained for cytokeratin 18, either through indirect detection using Thermo Scientific Pierce Biotin-Conjugated Goat Anti-Rabbit Secondary Antibody, Poly-HRP conjugate and Metal Enhanced DAB to develop the signal (top panel), or by directly biotinylating the primary antibody and using Thermo Scientific DyLight 594-Conjugated Streptavidin for fluorescent detection (red; right panel). Thermo Scientific Pierce Hoechst Stain was also used to fluorescently label cell nuclei (blue; bottom panel).

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 horseradish peroxidase (HRP) or alkaline phosphatase (AP), which is then activated by adding substrate. Alternatively, the primary antibody is conjugated to a fluorophore for detection by fluorescence microscopy. While direct detection does not require an additional step of adding a secondary antibody, signal amplification is not possible and therefore the signal may be difficult to detect if the protein is found in small quantities. Additionally, the conjugation process may in some cases interfere with the binding of the primary antibody with the target antigen. A major advantage of this method, though, is that multicolor fluorescent staining is only limited by the number of different fluorophores that the detection system (fluorescent microscope) can detect and not by the availability of the different primary antibodies in various species, which can often be restricted to just a single species.

Indirect Antibody Detection

The indirect method of detection employs a secondary antibody with specificity against the unlabeled primary antibody to amplify the primary 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.

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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 (top panel) or by indirect detection using a primary anti-cytokeratin 18 antibody and Thermo Scientific Biotin-Conjugated Goat Anti-Mouse Antibody (bottom panel). In both approaches, Thermo Scientific DyLight 649-Conjugated Streptavidin was used to fluorescently detect the target antigen.

Indirect Methods of Antigen Detection

Avidin is a tetrameric protein naturally found in egg whites 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.

The Labeled Streptavidin Biotin (LSAB) method builds upon the ABC method by conjugating the enzyme reporter directly to avidin. 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 8-fold over traditional ABC detection.

The Phosphatase-Anti-Phosphatase (PAP) method offers greater sensitivity because the approach uses an additional level of amplification over ABC and LAB. 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 is 100 to 1000 times greater than just secondary antibody amplification. An added benefit of PAP is that less primary antibody can be used for each sample staining, although it requires an additional antibody (anti-phosphatase).

Schematic of the indirect detection methods for IHC.


Immunoenzymatic tissue staining results from the reaction of a soluble substrate with an enzyme to produce an insoluble, colored product. The intensity of the color produced when the substrate is added should correlate with the concentration of the primary antibody and the respective tissue antigen. Many enzymes are used for these applications, but the most common selections are horseradish peroxidase (HRP) and calf intestinal alkaline phosphatase (AP). Enzymatic activity is dependent on several variables, including enzyme and substrate cooncentration, buffer, pH, temperature, and possibly light.

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.

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Substrates for immunohistochemistry

Substrate Format Enzyme Features Sensitivity Signal color
DAB Dry powder HRP Can be formulated Medium Brown
Metal-enhanced DAB 2-component reagent kit HRP 50X more sensitive than
DAB alone
Highest Brown to black
BCIP Dry powder AP Can be formulated Medium Blue to purple
NBT Dry powder AP and glucose oxidase Non-carcinogenic Medium Blue to purple
1-step NBT/BCIP Single step, ready-to-use AP Low background for high
High Black to purple
1-step NBT/BCIP + suppressor Single step, ready-to-use AP Contains levamisole for
endogenous peroxidase inhibition
High Black to purple

Optimization Strategies

Signal amplification can be further enhanced by tyramide signal amplification (TSA), which may be used in conjunction with peroxidase-conjugated detection systems. This approach utilizes the catalytic activity of the peroxidase conjugate to increase the amount of dye or label localized to the antigen-bound primary antibody. Briefly, after the primary and peroxidase-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 very low-expressed proteins by 100-fold, although downsides of this approach are the high cost of the proprietary kits, the high level of optimization required and the possible increase in background signal.

Avidin 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, while evolutionarily unrelated to avidin, shows a similar affinity for biotin but can reduce high background staining because it is not glycosylated and is only slightly negatively charged. Thermo Scientific NeutrAvidin Protein is a commercially-available form of avidin that is deglycosylated and modified to have a neutral charge to further reduce nonspecific binding and subsequent background staining.

Biotin-binding conjugates are normally limited to carrying 1-3 HRP molecules per protein in order to maintain enzymatic activity.Because of the small size of the HRP enzyme, 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 (picograms and femtograms) by increasing the molar ratio of HRP on the conjugate through proprietary techniques while maintaining enzymatic functionality.