Autostainer

EBV status was determined by Epstein-Barr virus-encoded Early RNA (EBER) in situ hybridization (ISH) on the samples, brushes or TMA. Ventana BenchMark automated staining instruments (Ventana Medical systems, Tuscon, AZ, USA) were used for ISH of the samples or TMA using an EBV-specific probe (INFORM EBER PROBE; Ventana Medical systems) and ISH iVIEW Blue detection kit (Ventana Medical systems, Inc.) for staining using the manufacturer’s instructions in Innsbruck, Utrecht, Hong Kong, Stanford and London. Shenzhen used an EBER Probe (Zhongshan Jinquaiao Biotechnology Co.; Beijing, China) and an autostainer (Ventana Medical Systems, Inc.) to perform ISH. Singapore used a BOND^TM Ready-to-use ISH EBER probe and a Leica Bond-Max autostainer (all Leica Biosystems, Wetzlar, Germany) for this purpose. In situ hybridization of xenografts and cell pellets was done using an EBV-specific probe (INFORM EBER PROBE; Ventana Medical systems) and ISH iVIEW Blue detection kit (Ventana Medical systems, Inc.) using the manufacturer’s instructions.

Histologic sections (5 μm) were obtained from 10% formalin-fixed paraffin-embedded (FFPE) blocks and stained with hematoxylin and eosin (H&E) for microscopic evaluation. Immunohistochemistry using a mouse anti-VDR monoclonal antibody (D-6: sc13133 Santacruz Biotechnology, Dallas, Texas, USA) and anti-FOXP3 (NB600–242 Novus biologicals, CO, USA) was performed on serial 5 μm -thick FFPE tissue sections on autostainer (Ventana Medical System, Tuscon, AZ) as per the manufacturer’s instructions. Nuclear and/or cytoplasmic (VDR) and nuclear (FOXP3) immunoreactivity was assessed in all cases. Scoring was done based on percentage of positive cells 0 (no staining), 1+ (< 10%), 2+ (10–50%), 3+ (> 50%) cells and semiquantitative data (0–3+) was used for correlation analysis. Cases were categorized as either focal/absent (0/1+) or diffuse (2+/3+).

Representative areas from formalin-fixed, paraffin-embedded tissues were subjected to IHC, which was performed on 4-μm-tick sections using a Ventana auto-stainer and an ultra-View DAB Detection Kit (Ventana, Tucson, Arizona), according to the manufacturer’s instructions. Primary antibody for p53 (clone DO-7, catalog No.M7001, DAKO, Denmark, Glostrup, 1:1000, Mouse monoclonal) was used. The p53 IHC staining were evaluated by two pathologists (JK and YNS) and classified into 3 categories: overexpression (strong diffuse nuclear immunoreactivity in all tumor cells), null (complete absence of nuclear immunoreactivity in all tumor cells), and usual (neither overexpression nor null, variable nuclear immunoreactivity in tumor cells).

HER2 protein expression in gastric and GEJ adenocarcinomas was evaluated by IHC on FFPE tissues, using clone 4B5 (rabbit monoclonal, Ventana) on a Ventana auto-stainer. Membrane staining of tumor cells is evaluated and graded as follows: 0 (negative), no immunoreactivity (biopsy) or membranous immunoreactivity in < 10% of tumor cells (resection); 1+ (negative) tumor cell cluster (5 cells) with faint immunoreactivity regardless of percentage of cells stained (biopsy) or faint staining in > 10% of tumor cells but only a portion of the membrane is positive (resection); 2+ (equivocal), tumor cell cluster with weak to moderate complete, basolateral or lateral membrane immunoreactivity regardless of percentage of cells (biopsy) or complete, basolateral or lateral membrane staining in > 10% of tumor cells (resection); 3+ (positive), tumor cell cluster with strong complete, basolateral or lateral membrane staining regardless of percentage of cells stained (biopsy) or strong complete, basolateral, or lateral membrane staining in > 10% of cells (resection). Equivocal (2+) staining triggers reflexive FISH testing.

Both the RA control and BPD mice were anesthetized (using an overdose of a cocktail of xylazine-ketamine) and lung and heart tissues were harvested after perfusion and fixed overnight in 4% paraformaldehyde. Fixed tissues were then washed in fresh PBS, and stored in 70% ethanol before being processed in an autostainer (Ventana, CA, USA) by the Histology Core Facility of Wistar Institute, Philadelphia to be stained with hematoxylin and eosin (H&E) for lung morphometry or immunohistochemistry/immunofluorescence as previously described [19 (link),23 (link),24 (link)]. Immunofluorescence staining was performed for Ki67 (Abcam, MA, USA 1:10) SP-C (Abcam, MA, USA, 1:100), RAGE (R&D Systems, Minneapolis, MN, USA, 1:100), and Von Wilebrand Factor (vWF-DAKO, 1:100) on lung paraffin sections following the protocol as described earlier [24 (link)], while TUNEL staining was performed following the manufacturer’s instructions (Roche Diagnostics, Indianapolis, IN, USA).