Anti rabbit hq detection system

Ki-67 IHC was performed at Johns Hopkins IHC core facility. Briefly, immunolabeling for Ki-67 was performed on formalin-fixed, paraffin-embedded sections on a Ventana Discovery Ultra autostainer (Roche Diagnostics). Following dewaxing and rehydration on board, epitope retrieval was performed using Ventana Ultra CC1 buffer (#6414575001, Roche Diagnostics) at 96°C for 48 minutes. Primary antibody anti-Ki-67 (1:200 dilution; #Ab16667, lot number GR3185488–1, Abcam) was applied at 36°C for 60 minutes. Primary antibodies were detected using an anti-rabbit HQ detection system (#7017936001 and 7017812001, Roche Diagnostics) followed by Chromomap DAB IHC detection kit (#5266645001, Roche Diagnostics), counterstaining with Mayer’s hematoxylin, dehydration, and mounting. Whole slide imaging was performed at the Oncology Tissue Services Core of Johns Hopkins University. Scanning was carried out at up to 40X magnification (0.23 microns/pixel) using a Hamamatsu Nanozoomer S210 digital slide scanner (Hamamatsu Photonics, Shizuoka, Japan). Whole slide images were visualized in Concentriq digital pathology platform (Proscia, Philadelphia, PA), and representative images at 20X magnification were shown.

Immunostaining was performed at the Oncology Tissue Services Core of Johns Hopkins University School of Medicine. Immunolabeling for pSTAT3 was performed on formalin-fixed, paraffin-embedded sections on a Ventana Discovery Ultra autostainer (Roche Diagnostics). Briefly, following dewaxing and rehydration on board, epitope retrieval was performed using Ventana Ultra CC1 buffer (catalog no. 6414575001, Roche Diagnostics) at 96°C for 64 min. Primary antibody, anti-pSTAT3 (1:100 dilution; catalog no. 9145L, lot number 22, Cell Signaling Technology), was applied at 36°C for 60 min. Primary antibodies were detected using an anti-rabbit HQ detection system (catalog nos. 7017936001 and 7017812001, Roche Diagnostics), followed by ChromoMap DAB IHC detection kit (catalog no. 5266645001, Roche Diagnostics), counterstaining with Mayer’s hematoxylin, dehydration, and mounting.

Immunostaining was performed at the Oncology Tissue Services Core of Johns Hopkins University School of Medicine. Immunolabeling for pMLC2 was performed on formalin-fixed, paraffin-embedded sections on a Ventana Discovery Ultra autostainer (Roche Diagnostics). Briefly, following dewaxing and rehydration on board, epitope retrieval was performed using Ventana Ultra CC1 buffer (catalog no. 6414575001, Roche Diagnostics) at 96°C for 48 min. Primary antibody, pMLC2 (1:50 dilution; catalog no. 3671, lot number***, Cell Signaling Technology), was applied at 36°C for 60 min. Primary antibodies were detected using an anti-rabbit HQ detection system (catalog nos. 7017936001 and 7017812001, Roche Diagnostics), followed by ChromoMap DAB IHC detection kit (catalog no. 5266645001, Roche Diagnostics), counterstaining with Mayer’s hematoxylin, dehydration, and mounting.