Immunohistochemical Analysis of Formalin-Fixed Tissues

Formalin-fixed, paraffin-embedded tissues were sectioned and stained with hematoxylin and eosin (H&E) using standard methods. The primary antibodies, concentration and specific pretreatments are summarized in Supplementary Table S1. Briefly, serial sections were incubated with the primary antibody overnight at 4 °C and then rinsed and stained using the ABC kit (Vector Laboratories, Burlingame, CA, USA) following the manufacturer’s instructions. Slides were developed in DAB developed in DAB (Invitrogen, Camarillo, CA, USA) and counterstained with hematoxylin. Quantitative IHC was performed on slide captures representing 4–10 representative regions per sample and percent positivity or H scores were calculated with the QuPath v0.2.0-m4 image analysis software [37 (link)].