Immunohistochemistry (IHC) combines anatomical, immunological, and biochemical techniques to image discrete components in tissues by using appropriately labeled antibodies to bind specifically to their target antigens in situ. IHC makes it possible to visualize and document the high-resolution distribution and localization of specific cellular components within cells and within their proper histological context. Detecting the target antigen with antibodies is a multi-step process that requires optimization at every level to maximize signal detection.

IHC target antigens are detected directly through either chromogenic or fluorescent means, and the type of readout depends on the experimental design. Chromogenic detection is based on antibodies conjugated to enzymes where the staining process exploits enzymes which catalyze the deposition of a colored staining product at antigenic sites within the sample. Most often, the enzymes used are horseradish peroxidase (HRP) or alkaline phosphatase (AP), which are conjugated to primary or secondary antibodies. IHC can also employ fluorescent means of detection. This procedure is accomplished either by using fluorophore-conjugated primary antibodies targeting a specific protein, or by first labeling with primary antibodies followed by secondary antibody detection.

All Antibodies for use in IHC

Considerations when choosing an IHC antibody

  1. Whether it will work in IHC. Not all antibodies work for all applications. If the antibody has been validated* for IHC, then the manufacturer’s guidelines or any published references can be used as guidance for the experiment. Other methods that require the antibody to recognize the target in a native state, such as immunofluorescence (IF) and immunocytochemistry (ICC), are also good indicators.
  2. Whether it is specific. Specificity of the antibody has been a growing and understandable concern. Antibody specificity becomes more important when studying post-translational modifications or specific isoforms of the protein. Invitrogen antibodies undergo a two-part testing approach: functional application validation and targeted specificity verification. Functional application validation provides information on whether the antibody works in the said application. Target specificity verification ensures the antibody is recognizing the target protein of interest.
  3. In case of fluorescent detection, selection of primary antibody based on host. More than one protein can be detected through multiplexing–this allows for target co-localization studies can be carried out. Primary antibodies from different species must be used to prevent secondary antibody cross-reactivity and improve specificity.

Both monoclonal and polyclonal antibodies can work in IHC. The key requirement is that the specific epitope of interest be exposed. One of the advantages of using a monoclonal antibody is that generally it is more specific, but this is associated with a higher likelihood that the one epitope it recognizes is buried. Unless monoclonal antibodies are specifically screened or designed for use in IHC, polyclonal antibodies would be a good alternative. Polyclonal antibodies recognize multiple epitopes of the targets, but at the same time are more likely to be cross-reactive.

IHC data examples

Each Invitrogen antibody that is indicated for use in IHC applications has undergone functional application testing. Here are some examples of that testing.

Detection of claudin 5 in kidney tissue. Immunohistochemistry analysis of claudin 5 showing staining in the membrane and cytoplasm of paraffin-embedded human kidney tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 minutes. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 minutes at room temperature, washed with ddH2O and PBS, and then probed with a Claudin 5 Mouse Monoclonal Antibody (Cat. No. 35-2500) diluted in 3% BSA-PBS at a dilution of 1:20 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

IHC of Cardiac Troponin T in mouse heart tissue. Frozen sections were fixed with 4% paraformaldehyde for 20 minutes, permeabilized with 0.1% Triton X-100 for 15 minutes and blocked with 10% normal goat serum for 1 hour at room temperature. Whole heart longitudinal sections were then incubated with Cardiac Troponin T Mouse Monoclonal Antibody (Cat. No. MA5-12960, 5µg/mL) overnight at 4°C, followed by Goat anti-Mouse IgG (H+L) Superclonal Secondary Antibody, Alexa Fluor 488 conjugate (Cat. No. A28175, 1:2000,45 mins). Nuclei (blue) were stained using SlowFade Gold Antifade Mountant with DAPI (Cat. No. S36938), and cytoskeletal F-actin (red) was stained using Rhodamine Phalloidin (Cat. No. R415, 1:300). Panel a) represents staining with the matched isotype control. Panel b) shows representative sections stained for Cardiac Troponin T (green). The images were captured at 20X magnification.

For Research Use Only. Not for use in diagnostic procedures.