A sensitive two-step HRP and AP detection method
Biocare’s 4plus detection systems are designed for reliable, cost-effective, two-step detection, providing a high-level of sensitivity. The vast majority of primary antibodies can be diluted two-fold compared to other commercially available avidin-biotin detection systems. Biocare’s prediluted antibodies are all standardized to work with the 4plus detection system.
4plus detection system can be used with mouse and rabbit primary antibodies. After labeling the antigen with primary antibody, a species-specific or universal, affinity-purified, biotinylated secondary antibody is added to bind. Horseradish peroxidase (HRP) or alkaline phosphatase (AP) labeled-streptavidin is then added. A chromogen/substrate is applied that reacts with a HRP or AP to produce an intense color signal.
Mouse Smooth Muscle Actin with B- and T-cells
Biocare’s Avidin-Biotin Blocking Kits are optimized for blocking endogenous biotin in tissue sections. Endogenous biotin is abundant in cells such as hepatocytes, kidney tubules and many tumors, particularly carcinomas. Biocare’s biotin-blocking kits can eliminate nonspecific false-positive staining when using avidin-biotin detection systems. The blocking step is particularly critical when using heat-induced epitope retrieval (HIER), which unmasks endogeneous biotin in tissues. (CAP checklist question ANP.22615)
Used with both mouse and rabbit primary antibodies. 4plus Universal HRP and ALP and 4plus species specific components are also available as bulk reagents.
4-plus Detection Kits
|4plus HRP Universal Detection|
4 Plus Biotinylated Secondary Antibodies
|Universal Goat Link|
|Goat Anti-Mouse IgG|
|Goat Anti-Rabbit IgG|
|Goat Anti-Rabbit IgG|
|Goat Anti-Rat IgG|
|Horse Anti-Mouse IgG|
4 Plus Streptavidin-Enzyme Conjugates
|4plus Streptavidin AP Label|
|4plus Streptavidin HRP Label|
Mouse-on-Mouse Avidin-Biotin Detection
Mouse monoclonal antibodies can be used for immunohistochemical detection on mouse tissues; however, the biotinylated secondary antibody used for detection of the primary antibody will also bind to the endogenous mouse IgG in the tissue, resulting in background staining from bound-mouse IgG. To circumvent the problem, Biocare has developed the MM Biotinylation Kit. The kit provides all the reagents necessary to biotinylate a primary antibody.
The basic concept of the kit is very simple. The antibody is mixed with a biotinylation reagent for 30 minutes prior to the application of primary antibody. As little as 5 to 10 µL of antibody can be used. Once the complex is formed, a mopping reagent is added and then the biotinylated antibody complex is applied to the tissue specimen. The tissue is then incubated with the strepavidin-peroxidase reagent, and the color reaction is developed by a DAB chromogen substrate.
The system has been specially formulated to eliminate endogenous mouse IgG background staining. Even the most difficult tissues such as lymph, spleen, lung and kidney are virtually background free.
Mouse-on-Mouse Biotinylation: Mouse Smooth Muscle Actin with B- & T-cells
|MM Biotinylation Kit|