Abstract 3860: Rodent multispecies multiplex immunohistochemistry using digoxigenin and polymer detection methods in mouse tissues
Authors: Vargas Joseph , Tacha David , Figueroa Sara , Douglas Cristin 
Published: August 2018

Abstract
When mouse, rat, and rabbit antibodies are used, consideration of the target tissue is of critical importance. Endogenous IgGs present in tissues pose significant challenges to interpretation. When a species conflict exists for the antibody and the target tissue, it is likely to generate undesired background staining due to the detection system. In the past, mouse-on-mouse and rat-on-mouse utilized either a biotinylated primary antibody or a biotinylation approach to label the antibody. Tissues rich in endogenous biotin such as kidney or liver were difficult to use in these systems as streptavidin-horseradish peroxidase (HRP) or anti-biotin techniques were used as the detection complement. Attempts to negate endogenous biotin were minimally effective as avidin-biotin blocking steps were unlikely to work acceptably in tissues such as kidney and liver. Using anti-digoxigenin (dig) and polymer technology to generate multi-species multiplex stains may provide a solution.
Materials and Methods: Mouse tissues were fixed for 24 hours and embedded in paraffin. Sections were cut at 5 microns and dried on slides before deparaffinizing and peroxidase blocking. Slides were then heated at 110°C for 15 minutes (spleen, liver, kidney) or 80°C for 60 minutes (brain) in a citrate-based buffer (pH 6.2) prior to application of antibodies. A sequential approach of two applications of dig-labeled mouse-on-mouse (MM) detection was performed with PAX8 (M) and CD10 (M) as well as with Neurofilament (M) and GFAP (M). Anti-rat alkaline phosphatase and anti-rat HRP polymer sequential applications were also executed with rat monoclonals CD4 and CD8. Then, a multispecies sequential multiplex stain using MM anti-dig along with anti-rabbit polymer was achieved with mouse hepatocyte specific antigen and rabbit CD3. Finally, a four-step cocktailed multispecies multiplex was developed using a rat monoclonal F480 combined with a rabbit CD3 in conjunction with a cocktail of anti-rat polymer and anti-rabbit polymer. Visualization in each assay was achieved by the use of two chromogenic end-points. Slides were counterstained in either hematoxylin or methyl green prior to dehydration and mounting.
Results: Rodent multiplex stains were easily achieved with dig-based MM and cocktailed methods that use mouse/rabbit or rat/rabbit approaches. Specific, clean, and background-free staining on spleen, liver, kidney and brain mouse tissues was achieved with excellent signal-to-noise in both sequential and cocktailed formats.
Conclusion: Using mouse-on-mouse anti-digoxigenin technology in multiplex staining is a superior approach to streptavidin or biotin strategies in rodent systems. Additional multiplexing techniques with rat or rabbit antibodies using polymer-based detections generates excellent staining with a significant reduction of background and an exceptional signal-to-noise ratio in mouse tissues.
Citation Format: Joseph Vargas, David Tacha, Sara Figueroa, Cristin Douglas. Rodent multispecies multiplex immunohistochemistry using digoxigenin and polymer detection methods in mouse tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3860.