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Tips and hints for your experiments
In an immunohistochemistry (IHC) experiment a primary antibody binds specifically to a protein of interest present in a tissue. The antibody binding is then visualized by a detection system, which provides information about if and where the protein is present in the tissue. IHC is a common method in diagnostics to determine morphological abnormalities and the presence of biomarkers indicative of certain diseases such as cancer.
Since the first IHC experiments in the 1940s much progress has been made in terms of developing different and increasingly sensitive detection systems. In the beginning almost exclusively secondary antibodies conjugated to enzymatic labels were used for visualization, while these days kits, directly conjugated primary antibodies and fluorescent labels are increasing in popularity. This trend is due to researchers wanting to detect several antigens simultaneously in one tissue specimen. This process is known as multiplexing and has many advantages, including cost and time savings and increased sample characterization.
Tips and hints for your IHC-paraffin experiments
Before starting the experiment, make sure you have selected an antibody that has been validated in IHC-P, that you have included controls in your experimental design, and that your microscope is set up to detect the antibody staining.
View a list of all IHC-paraffin tested antibodies.
Detailed protocols for IHC staining of frozen and paraffin embedded tissues can be found here.
Antigens can be masked as a result of the fixation process, which makes antibody binding impossible. The unmasking can be reversed with a technique called antigen retrieval/antigen unmasking, which is either mediated by heat (HIER; heat-induced epitope retrieval) or proteases (PIER; proteolytic-induced epitope retrieval). The latter uses enzymes such as proteinase K, trypsin and pepsin. The PIER method acts by degrading the peptides masking the epitope. However, PIER might also result in alterations to the specimen morphology or the antigen itself. Consequently, PIER is less frequently used than HIER, which acts by restoring the secondary and tertiary structure of an epitope.
In order to establish whether antigen retrieval should be performed and by which method the following guidelines should be followed:
To avoid staining artifacts it is important:
Blocking should be performed prior to incubation with the primary antibody to prevent non-specific antibody binding.
View all Bio-Rad sera here.
View all secondary antibodies
all avidin/streptavidin products
In immunohistochemistry the enzymatic labels horseradish peroxidase (HRP) and alkaline phosphatase (AP) are mainly used.
In an immunoenzymatic staining a colored precipitate is formed due to the reaction of an enzyme with its substrate. This reaction converts a chemical compound called chromogen into the precipitate (see below formula; reference (1)).
Factors to consider:
The most popular chromogens for both HRP and AP and the resulting precipitate colors are shown below (adapted from (1)).
(1) Agilent Technologies (2009), Education Guide: Immunohistochemical staining methods edition 5.
Available here (Accessed: January 02, 2015)
Like controls, counterstaining is crucial for every IHC experiment, as the counterstain provides background contrast and puts the observed staining into perspective (e.g. by visualizing nuclei).
The most commonly used chromogenic and fluorescent counterstains are shown below (adapted from (1)).
(1)Paul, C., Counterstaining for Immunohistochemistry: Choices, Choices
Available here (Accessed: December 02, 2015).
Mounting media are essential for making permanent slides and assist in making the coverslip adhere to the slide. Mounting has the purpose of protecting the specimen from damage while adding contrast during microscopy. Two types of mounting media exist:
Mounting media can be further differentiated into media that solidify or stay liquid (2). In general organic solvent based media solidify, while aqueous ones remain in a liquid state.
Bio-Rad offers permanent aqueous mounting medium. This mounting medium is suitable for use with peroxidase and alkaline phosphatase chromogens, Fast red, BCIP/NBT, BCIP/INT, Aminoethylycarbazole (AEC) and DAB/DAB with cobalt and nickel chromogens, and is also compatible Nuclear fast red (NFR) counterstains.
(1) Renshaw, S. (editor) (2007) Imunohistochemistry. Methods Express Scion Publishing
(2) Kim, O., MicrobeHunter Microscopy Magazine, An overview of mounting media for microscopy. Available here (Accessed: July 15, 2014).
(3) North, A.J. (2006) Seeing is believing? A beginners' guide to practical pitfalls in image acquisition. JCB vol. 17 no. 1 9-18.
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