Dab kit

For p16 immunohistochemistry (IHC), staining was performed with p16 E6H4 (Roche) according to the manufacturer’s protocol on a BenchMark ULTRA IHC/ISH Staining Module. The slides were deparaffinated at 72 °C with EZ-Prep (Ventana). Slides were then pretreated with Cell Conditioner 1 (Ventana) followed by Cell Conditioner 1 ULTRA CC1 protocol. Slides were subsequently incubated with p16 antibody for 20 min. Staining was performed with DAB Kit (Ventana). Counterstaining was performed with Hematoxylin (Ventana) and Bluing Reagent (Ventana).
HPV in situ hybridization (ISH) was performed using INFORM HPV II Family 6 (Roche, 800–2220) for HPV low risk, and INFORM HPV III Family 16 (Roche, 800-4295) for HPV high-risk strains. Staining of HPV ISH was carried out with Iview Blue Plus Detection Kit (Roche, 760-097) according to protocol. HPV III Family 16 detects HPV high risk strains 16, 18, 31, 33, 35, 45, 52, 56, 68 and 66. HPV II Family 6 detects low-risk strains and does not hybridize high-risk strains. Red Counterstain II (Ventana) was used for counterstaining.
A trained uropathologist evaluated the slides.

IHC staining was performed using the Discovery Ultra autostainer (Ventana, Roche, Tucson, AZ) on formalin-fixed paraffin-embedded whole tissue sections. The slides were deparaffinized in the instrument (68°C, 3 cycles á 12 min). Antigen retrieval was applied using ULTRA Cell Conditioning-1 (Ventana, Roche) for 32 minutes at 95°C. The sections were incubated for 32 minutes with IVD primary mouse monoclonal antibody anti-CD34 (clone QBEnd/10; cat# 790-2927; Ventana, Roche). As a secondary antibody, OmniMap anti-Mouse HRP (cat# 760-4310, Ventana, Roche) was loaded for 16 minutes, followed by 4 minutes of HQ HRP amplification (cat# 760-052, Ventana, Roche). The detection chromogen used was the DAB kit (cat#760-159; Ventana, Roche) with 32 minutes of incubation. Counterstaining was performed using hematoxylin II (cat#790-2208, Ventana, Roche) for 12 minutes and then loading bluing reagent for 4 minutes.
Slides were digitized using a Pannoramic 250 Flash III (3DHistech, Budapest, Hungary), slide scanner.

Formalin-fixed, paraffin-embedded (FFPE) tissue microarrays (TMA) composed of meningioma tumor probes were cut in 2 µm sections and stained using the BOND Fully Automated IHC Staining System or Ventana Roche BenchMark ULTRA Fully Automated IHC Staining System. The sections were incubated with primary antibodies against CD4 (Ventana-Roche, Order No. 790-4423/05552737001, prediluted) and CD8 (DAKO A/S, Order No. M71030, dilution: 1:100). For the detection of T regulatory cells, antibodies were first applied against FOXP3 (Abcam Limited, Order No. ab2003, dilution 1:150) and then against CD3 (Thermo Scientific, Order no. MA1-90,582, dilution: 1:100). Visualization of the antibodies was performed with the VENTANA BenchMark ULTRA, the DF AP or DAB kit. All sections were counterstained with hematoxylin.