The Immunohistochemistry Detection Kit, HRP, Mouse, Rat, BioAssay(TM) kit is designed to allow immunohistochemical studies of mouse or rat samples using mouse, rabbit, rat and guinea pig primary antibodies. The kit can be used with all common sample preparation methods including: frozen tissues, paraffin-embedded sections, and cell smears. Unlike most immunostaining systems, in which the anti-mouse secondary antibody causes non-specific staining of endogenous rodent immunoglobulins, the kit uses the a specific method to prevent this unwanted staining. The kit incorporates with a high sensitive horseradish peroxidase (HRP) second antibody to increase sensitivity. And the detection system is faster and cleaner because it uses one step of HRP conjugated to second antibody instead of two steps, as in the LAB-SA method.
Immunohistochemistry Detection Kit, HRP, Mouse, Rat, BioAssay(TM) does not contain biotin or streptavidin, so potential background due to endogenous biotin activity is completely avoided. In addition, a special blocking system is used to eliminate background staining. After a user-supplied primary antibody is incubated with the sample, a HRP-conjugated, affinity-purified secondary antibody is bound to the primary antibody. A DAB chromogen/substrate system then creates an intense brown deposit around the antigen/antibody/enzyme complex in the sample.
The kit uses a horseradish peroxidase-conjugated second antibody, and a substrate-chromogen mixture to demonstrate antigen in cells or tissues.
The principle of this staining system is as follows:
1) Paraffin embedded tissue should be deparaffinized and rehydrated.
Endogenous peroxidase activity can be quenched by treating tissue sections with 3% hydrogen peroxide in absolute methanol.
2) Non-specific background is eliminated by incubating the tissue section with blocking solution A (I7506-05A), and blocking solution B (I7506-05B).
3) The mouse, rabbit, rat or guinea pig primary antibody, for a specific antigen, is incubated on the tissue.
4) Incubation is followed by addition of a HRP-conjugated second antibody (I7506-05C), binding to the primary antibody.
5) The presence of peroxidase can be revealed by addition of substrate chromogen solution (I7506-05D, I7506-05E & I7506-05F: see protocol).
Peroxidase will catalyze the substrate (hydrogen peroxide) and convert the chromogen (DAB) to a brown deposit, which demostrates the location of the antigen.
The kit is designed to reveal antigens on mouse or rat tissue or cell samples that have reacted with a user-supplied, primary antibody. Depending on the reactivity of the primary antibody, antigens such as cell surface markers, hormones, cytoskeletal proteins and tumor markers can be detected on properly prepared samples. Frozen or paraffin-embedded tissues, freshly prepared lymphocytes, and fixed cultured cells are the most common preparations. Use for research only.
I7506-05A: Blocking Solution A, 1x6ml (ready-to-use).
I7506-05B: Blocking Solution B, 1x6ml (ready-to-use).
I7506-05C: Secondary Antibody (HRP), 6ml (ready-to-use).
I7506-05D: Substrate Buffer (20X), 1x2ml
I7506-05E: DAB Chromogen (20X), 1x2ml
I7506-05F: 0.6% H2O2 (20X), 1x2ml
I7506-05G: Hematoxylin Solution, 1x6ml (ready-to-use)
I7506-05H: Mounting Solution, 1x10ml (ready-to-use)
Storage and Stability:
May be stored at 4 degrees C. Do not freeze. Stable for 6 months. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.