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.
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.
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.
*The use or any variation of the word “validation” refers only to research use antibodies that were subject to functional testing to confirm that the antibody can be used with the research techniques indicated. It does not ensure that the product(s) was validated for clinical or diagnostic uses.
For Research Use Only. Not for use in diagnostic procedures.