Biocare Medical Mouse-on-Mouse Specialty Detection

Mouse on Mouse Biotinylation Double Stain Kit
Biotinylation involves the tagging of a mouse primary antibody with biotin. One of the drawbacks of these kits is that certain tissues (e.g. kidney, spleen, colon and liver) may require an avidin-biotin blocking step to eliminate endogenouse biotin. This procedure may add as much as one hour to the staining time. If a double stain is to be performed using a mouse on mouse protocol, a sequential protocol using an elution step is performed. Depending on the sensitivity of the detection kit, a mouse on mouse double stain procedure could take up to four to six hours. To circumvent these problems, a new sequential 5-step mouse on mouse double stain protocol has been developed that incorporates avidin into a protein blocking reagent and biotin into antibody diluent. A biotinylated mouse primary antibody is cocktailed with a rabbit antibody. Next, a cocktail of streptavidin-HRP and a biotin-free goat anti-rabbit alkaline phosphatase (AP) is applied. DAB and Fast Red were used sequentially for two-color staining. Double staining was easily achieved in five major sequential steps in approximately 2.2 hours, eliminating an avidin-biotin blocking step, elution step, and sequential staining steps.
Cat. No. MM519 G, H 6.0, 25 ml MM Double Stain Kit

Components Available For Mouse on Mouse IHC

Mouse-on-Mouse Biotinylation Kit
Mouse monoclonal antibodies can be used for immunohistochemical detection on mouse tissues; however, the biotinylated secondary antibody used for detection will bind to the primary antibody and 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 UL 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 streptavidin-peroxidase, 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 tissues are virtually background free.
Cat. No. MMBK, G, H, available in 6 and 25 ml

(Left) Mouse Anti-PCNA on mouse spleen.
(Right) Mouse Anti-Actin on mouse pancreas.

Vulcan Fast Red Chromogen Kit 2
Fast Red is a widely used chromogen for immunohistochemical staining. When in the presence of alkaline phosphatase (AP) enzyme, Vulcan Fast Red produces a bright fuchsin-red precipitate that is insoluble in organic solvents---and can be coverslipped with a permanent mounting media. This product is available in a stable two-component system, consisting of Vulcan Fast Red Chromogen and Vulcan Buffer.

MM Double Stain Detection
The MM Double Stain Detection reagent consists of a cocktail of Streptavidin-HRP and biotin-free conjugated goat anti-rabbit polymer alkaline phosphatase (AP) secondary antibodies. The conjugated goat anti-rabbit polymer alkaline phosphatase (AP) secondary antibodies react with both heavy and light chains on rabbit IgG. The new and innovative ALP-polymerization technology provides a significant increase in staining sensitivity when compared to other conventional AP-conjugated secondary antibodies. It is specially designed for a rapid double stain procedure. An antibody cocktail with a biotinylated mouse monoclonal and a rabbit polyclonal/monoclonal must be used. The overall staining procedure can be done with 5 major steps and in approximately 2.2 hours. It can be used manually or used with automated stainers.