DAB (3,30-diaminobenzidine) single-plex IHC detection was carried out initially to determine the appropriate primary antibody conditions using the Ventana Discovery Ultra automated IHC/ISH research platform (Roche Diagnostics Limited, Burgess Hill, UK) before translating to the TSA immunofluorescence.
Freshly cut 4 μm tissue sections were mounted on glass slides. Sections were placed in the Ventana Discovery Ultra and dewaxed; heat induced epitope retrieval was performed using CC1 reagent for 64 min. Primary antibodies were added by manual application and the final concentrations determined are provided in Table1 . Secondary HRP complexes and DAB were completed in a fully automated manner. Slides were washed, dehydrated through a series of alcohols and coverslipped. Slides were imaged using an Aperio CS2 whole slide scanner.
Freshly cut 4 μm tissue sections were mounted on glass slides. Sections were placed in the Ventana Discovery Ultra and dewaxed; heat induced epitope retrieval was performed using CC1 reagent for 64 min. Primary antibodies were added by manual application and the final concentrations determined are provided in Table
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