Anti rabbit hq

Immunohistochemistry was performed on TMA sections with the automated immunohistochemistry staining platform DISCOVERY ULTRA (Ventana Medical Systems, Inc.). Antigen retrieval was conducted with Cell Conditioning 1 (CC1) (Ventana) at 95 °C for 64 min. Slides were incubated with a 1:200 dilution of Abi1 antibody (Cell Signaling Technologies, 39444S) at room temperature for 2 h. For detection, a DISCOVERY ChromoMap DAB Kit, anti-HQ HRP, and anti-rabbit HQ (Ventana) were used. Digital images of stained TMAs were acquired with an SCN400 Slide Scanner (Leica Microsystems). Positively stained cells were analyzed with Aperio ImageScope (Leica Biosystems).

Immunohistochemistry of the FFPE tumor samples was performed on a BenchMark Ultra autostainer (TLE3) or Discovery Ultra autostainer (Glucocorticoid Receptor). Briefly, paraffin sections were cut at 3 µm, heated at 75°C for 28 min and deparaffinized in the instrument with EZ prep solution (Ventana Medical Systems). Heat-induced antigen retrieval was carried out using Cell Conditioning 1 (CC1, Ventana Medical Systems) for 64 min at 95°C.Glucocorticoid Receptor clone D6H2L (Cell Signaling) was detected using 1/600 dilution, 1 hr at 37⁰C and TLE3 using clone CL3573 (1/250 dilution, 1 hr at RT). Bound TLE3 was detected using the OptiView DAB Detection Kit (Ventana Medical Systems). Glucocorticoid Receptor bound antibody was visualized using Anti-Rabbit HQ (Ventana Medical systems) for 12 min at 37⁰C, Anti-HQ HRP (Ventana Medical systems) for 12 min at 37°C, followed by ChromoMap DAB Detection Kit (Ventana Medical Systems). Slides were counterstained with Hematoxylin and Bluing Reagent (Ventana Medical Systems).

Details on mutational analysis and immunohistochemical (IHC) staining for PD-L1 expression and CD8 infiltration was previously reported [9 (link)]. Double staining CD3 (yellow) followed by CD56 (purple) of whole slide sections prepared from FFPE resection specimens was performed on a Discovery Ultra autostainer. Slides were deparaffinised in the instrument and heat-induced antigen retrieval was carried out using Cell Conditioning 1 (CC1, Ventana Medical Systems) for 32 min at 95 °C. The CD3 was detected in the first sequence using clone SP7 (1/100 dilution, 32 min at 37 °C, ThermoScientific). CD3 bound antibody was visualized using Anti-Rabbit NP (Ventana Medical systems) for 12 min at 37 °C followed by Anti-NP AP (Ventana Medical systems) for 12 min at 37 °C, followed by the Discovery Yellow detection kit (Ventana Medical Systems). In the second sequence of the double staining procedure CD56 was detected using clone MRQ-42 (1:2000 dilution, 32 min at 37 °C, Cell Marque). CD56 was visualized using Anti-Rabbit HQ (Ventana Medical systems) for 12 min at 37 °C followed by Anti-HQ HRP (Ventana Medical systems) for 12 min at 37 °C, followed by the Discovery Purple Detection Kit (Ventana Medical Systems). Slides were counterstained with Hematoxylin and Bluing Reagent (Ventana Medical Systems).