Abstract 3831: The development of an immunohistochemical digoxigenin-labeled mouse on mouse single and double stain methodology
Authors: Vargas Joseph , Tacha David , Figueroa Sara 
Published: July 2017

Abstract
Introduction
When a mouse or monoclonal antibody is desired for immunohistochemical detection on mouse tissues, the anti-mouse secondary antibody will also bind to endogenous mouse IgG in the tissue and may produce unwanted background staining. In the past, mouse-on-mouse (MM) detection utilized a biotinylated primary antibody or biotinylation kit. Tissues rich in endogenous biotin such as kidney or liver made it difficult to use, as streptavidin-horseradish peroxidase is required for detection. Attempts to completely negate endogenous biotin are ineffective as avidin-biotin blocking steps are unlikely to work acceptably in problematic tissues. Therefore, the design of a digoxigenin-labeled detection system is advantageous for MM detection as it would eliminate these issues. This technology has broad application as it allows the use of a variety of monoclonal antibodies reactive on mouse tissues.
Materials and Methods
Mouse tissues were formalin-fixed for 24 hours and embedded in paraffin (FFPE). Sections were cut and dried on slides before deparaffinizing and peroxidase blocking in the usual manner. Slides were then HIER in a citrate-based buffer prior to application of antibody. Mouse antibody concentrates were selected and mixed with a Digoxigenin Anti-Mouse Linker and incubated at room temperature (RT) for 1 hour. An Absorption Reagent is then added to the mixture, and further incubated for 30 minutes. The mixture is then diluted in antibody diluent to the desired concentration and applied to the mouse tissue for 60 minutes at RT, followed by a rabbit anti-digoxigenin antibody for 15 minutes. Detection of anti-digoxigenin is performed with an incubation in either a goat anti-rabbit HRP or AP Polymer for 30 minutes. Visualization is accomplished by using a DAB or Fast Red chromogen. For double stains, a sequential DS method using two monoclonal antibodies was employed using a heating step in retrieval solution after the first chromogenic reaction followed by a second monoclonal antibody and detection with either a rabbit-AP or HRP polymer. Visualization of the second antibody target is accomplished by using Fast Red or a blue chromogen. Slides were coverslipped with a permanent mounting medium and evaluated using brightfield microscopy.
Results
MM single stains with a variety of antibodies were easily achieved with specific, clean and biotin-free staining. Sequential DS techniques using monoclonal antibodies were also achieved and visualized with DAB, Fast Red or blue chromogens. Both manual and automated procedures were used.
Conclusion
The replacement of biotin-labeled MM technology with a digoxigenin MM technology is a superior method that not only eliminates endogenous biotin, but allows utilization of HRP and AP polymers for double stain technology.
Citation Format: Joseph Vargas, David Tacha, Sara Figueroa. The development of an immunohistochemical digoxigenin-labeled mouse on mouse single and double stain methodology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3831. doi:10.1158/1538-7445.AM2017-3831