Discovery ultra automated immunostainer

Samples from cellular pellets and recovered tumors were fixed with 4% PFA for 4 h and then embedded into paraffin. After deparaffinization, eIF2α-phosphorylated staining was performed as follows. Sections of 5 μm were stained with a monoclonal anti-phospho-eIF2α (Ser51) (3597, Cell Signaling) on a Discovery Ultra automated immunostainer (Ventana, Tucson, USA). Antigen retrieval was performed by incubating slides in EDTA buffer (pH 8.0) for 32 min at 95°C. And then the antibody was incubated for 1 h at 37°C at the final concentration of 4 μg mL⁻¹. Finally, the samples were counterstain with hematoxylin II for 12 min followed by Bluing Reagent for 8 min (Ventana). After staining, images were acquired with a Virtual Slides microscope VS120-SL (Olympus, Tokyo, Japan), 20X air objective (0.75 NA).

Chromogenic immunohistochemical staining was performed using the Discovery Ultra Automated Immunostainer (Ventana Medical Systems, Tucson, AZ, USA) by the RI-MUHC Histopathology platform. Pre-treatment and post-treatment TMAs were stained for NETs and PMNs with H3Cit (Cat#: ab5103, 1:100, Abcam), NE (Clone: 950334, 1:100, Novus Biologicals, Oakville, ON, Canada) and CD8 (Clone: SP57, 1:100, Roche Diagnostics). TMAs were scanned using the Aperio Slide Scanner and slides were analyzed using QuPath software V6. TMAs were analyzed following the script created to analyze CD3 using fast cell counts^{67 (link)}.