Ventana discovery ultra staining module

To evaluate the transcriptional activity of HPV E6 and E7 and simultaneously obtain morphological information, the cases were submitted to chromogenic mRNA in‐situ hybridization (mRNA ISH). Tumor samples with an intact expression of the housekeeping gene cyclophilin B (PPIB) were included for E6/E7 mRNA ISH. A negative control (the bacterial gene diaminopimelate [DapB]) was also applied to determine non‐specific expression. The analysis was performed using the automated RNAscope VS Reagent Kit (Advanced Cell Diagnostics, Newark, CA, USA) on a Ventana Discovery ULTRA staining module (Ventana Medical Systems). We applied the high‐risk HPV18 probe covering the high‐risk genotypes 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, and 82 and the low‐risk HPV6 probe covering the HPV6, 11, 40, 42, 43, and 44 (Advanced Cell Diagnostics). A semi‐quantitative scoring algorithm recommended by the manufacturer was applied (grade 0: no staining or <1 dot per 10 cells; grade 1: 1–3 dots per cell; grade 2: 4–9 dots per cell; grade 3: 10–15 dots per cell and < 10% of dots were in clusters, grade 4: >15 dots/cell and > 10% of dots were in clusters) (Advanced Cell Diagnostics, 2017 ).

Immunohistochemistry was performed using a Ventana Discovery ULTRA Staining Module (Ventana Medical Systems, Tucson, AZ, United States). Formalin-fixed, paraffin embedded liver sections were stained with anti-CD68 (ab125212 Abcam, Cambridge, MA, United States), anti-collagen type 1 A1 (1310-01 Southern Biotech, Birmingham, AL, United States), or anti-catalase (PA5-29183, ThermoFisher).

Three 6-week-old treatment naïve C57BL/6 male mice were injected in the flank with 5×10⁵ HKP1, 3×10⁵ MC38 or 5×10⁵ LLC1 resuspended in 1:1 Matrigel:PBS. FFPE sections (4 µm) were prepared for each sample and subjected to routine H&E staining. Immunohistochemistry directed against alpha-CD8 (Abcam; clone: EPR21769; 1:500 dilution) was performed on FFPE slides on a Ventana BenchMark XT automated system. Steps were performed in line with the Ventana discovery ULTRA staining module (Roche Diagnostic). Briefly, deparaffinization was followed by cell conditioning using conditioner #1 at 95°C for 56 min and primary antibody incubation for 60 min. Then, one Drop of Omap anti-RB HRP (Roche Diagnostic) was applied with a 16 min incubation followed by incubation with one drop of hematoxylin (Roche Diagnostic) for 16 min. Finally, one drop of bluing reagent (Roche Diagnostic) was used for 4 min.