Bond intense r detection system

Chromogenic IHC was performed on formalin-fixed and paraffin-embedded (FFPE) tissue sections (5-µm thickness) using the Leica Bond III Autostainer system (Leica Biosystems, Deer Park, IL, USA) following the manufacturer’s instructions (25 (link)). The details of the primary antibodies used, their dilutions, and the procedure for antigen retrieval are summarized in Table 1. Slides were deparaffinized in Bond Dewax solution (Leica, AR9222) and hydrated in Bond Wash solution (Leica, AR9590). Heat-induced antigen retrieval was performed at 100 ℃ in either Bond-Epitope Retrieval Solution 1 pH 6.0 (Leica, AR9961) or Bond-Epitope Retrieval Solution 2 pH 9.0 (Leica, AR9640). Antigen retrieval was followed by a 5-minute peroxide blocking step (IPB5000L, Biocare Medical, Pacheco, CA, USA), after which slides were incubated with the primary antibody followed by Leica Post Primary and Novolink Polymer (Leica, RE7260-CE) secondary reagents. Antibody detection with 3,3'-diaminobenzidine (DAB) and hematoxylin counterstain was performed using the Bond Intense R detection system (Leica, DS9263). Stained slides were dehydrated and coverslipped with Cytoseal 60 (23-244256, Fisher Scientific, Pittsburgh, PA, USA). Appropriate positive controls were used for each assay.