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

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.