Catalyzed Signal Amplification (CSA) Method




CSA Methods From DakoCytomation:

1) CSA Systems
use Tyramide Signal Amplification. It is ideal for the following applications: i) Detecting small quantities of antigen; ii) Enhancing performance of low affinity mouse and rabbit antibodies; iii) Enabling compatibility of certain "tough" mouse and rabbit antibodies with paraffin embedded tissue sections. The simple protocol is as follows:
  1. Application of primary antibody
  2. Application of biotinylated linking antibody
  3. Application of the Tyramide Amplification Reagent
  4. Application of Streptavidin-HRP
  5. Application of the substrate chromogen
2) CSA II - Biotin-free Tyramide Signal Amplification System is a highly sensitive immunohistochemical (IHC) staining procedure incorporating a signal amplification method based on the peroxidase-catalyzed deposition of a fluorescein-labelled phenolic compound, followed by a secondary reaction with a peroxidase-conjugated anti-fluorescein. In the procedure, a mouse primary antibody is first detected with a peroxidase-conjugated secondary antibody. The next step utilizes the bound peroxidase to catalyze oxidation of a fluorescein-conjugated phenol (fluorescyl-tyramide) which then precipitates onto the specimen. The procedure is continued with detection of the bound fluorescein by a peroxidase-conjugated anti-fluorescein. Staining is completed using diaminobenzidine/hydrogen peroxide as chromogen/substrate, and can be observed with a light microscope.

This system is intended for use with primary antibodies from mouse supplied by the user for the qualitative identification of antigens by light microscopy in normal and pathological formalin-fixed, paraffin-embedded tissues.

In comparison to standard immunohistochemical methods, such as labelled streptavidin biotin (LSAB) or avidin-biotin complexes (ABC), tyramide amplification methods have been reported to be many fold more sensitive. The CSA II System is a simplified version of the extremely sensitive Catalyzed Signal Amplification System (code K1500) that utilizes biotinyl-tyramide. The highly sensitive CSA II System allows for the detection of very small quantities of target protein, as well as for the use of low affinity antibodies. This reagent system utilizes fluorescyl-tyramide, rather than biotinyl-tyramide, and does not contain avidin/biotin reagents, thus eliminating potential background staining due to reactivity with endogenous biotin.

Principles of Procedure:
  1. The specimens are first incubated with Peroxidase Block for five minutes to quench endogenous peroxidase activity.
  2. The specimens are then incubated for five minutes with a protein block to suppress nonspecific binding of subsequent reagents.
  3. Followed by a 15-minute incubation with an appropriately characterized and diluted mouse primary antibody or negative control reagent (user provided).
  4. This is followed by sequential 15-minute incubations with anti-mouse immunoglobulins-HRP, fluorescyl-tyramide hydrogen peroxide (amplification reagent) and anti-fluorescein-HRP.
  5. Staining is completed by a five-minute incubation with 3,3' diaminobenzidine tetrahydrochloride (DAB)/hydrogen peroxide, which results in a brown precipitate at the antigen site.