Bond dewax solution

IHC evaluations were performed on 2.5 μm sections of FFPE tissue mounted onto glass slides, dried, and baked at 60 °C for 30 min. Bond RX (Leica Biosystems) Immunostainer was used for automated staining. All slides were dewaxed in Bond dewax solution (product code AR9222, Leica Biosystems). Antigen retrieval was performed in Tris–EDTA buffer based (code AR9640, Leica Biosystems) for 30 min at 95° for anti-CD3 (1:400, Thermo Fisher MA190582); and in citrate buffer based (code AR9961, Leica Biosystems) for 30 min at 100° for anti-CD20 (1:200, Abcam, ab194970), anti-CD31 (1:30, Abcam ab28364), HLA-DR (1:400, Thermo Fisher MA532232;), anti-CD163 (1:400, Thermo Fisher PA578961) (Additional file 1: Table S1). All samples were then incubated with horseradish peroxidase-polymer for 15 min and subsequently visualized using 3,3-diaminobenzidine (DAB) as brown chromogen (Bond polymer refine detection, Leica Biosystems, Ref DS9800) for 10 min. Following these procedures, samples were counterstained with haematoxylin for 5 min, dehydrated, mounted on Pertex (Sakura).

Procedures examining the expression of p16 protein were carried out using a Bond-III Automated IHC/ISH Stainer (Leica Biosystem, Wetzlar, Germany) according to manufacturer’s instruction and reagents. Briefly, where available, 4 μm FFPE sections were mounted on glass microscope slides coated with Poly-L-Lysine. They were deparaffinized using Bond Dewax Solution (Leica Biosystem), rehydrated, and washed with Bond Wash Solution. The slides were incubated with Bond Epitope Retrieval Solution and heated at 100 °C for 20 min, washed, and Peroxide Wash Solution applied for 5 min. The p16 primary antibody (mouse monoclonal Anti-p16INK4a (E6H4), Ventana, Tucson, AZ, USA) was added on the slides for 15 min, followed by the anti-mouse secondary antibody (Post Primary Rabbit anti mouse IgG, ProClin, Leica Biosystem) for 8 min and the Bond Polymer Refine Detection solutions with intermittent washing. Slides were counterstained with Hematoxylin, and then dehydrated and mounted with DPX by using a Tissue-Tek film coverslipper (Sakura Finetek, Tokyo, Japan). Negative controls were obtained by excluding the primary antibody. Scoring of p16 IHC cytoplasmic and nucleic staining were evaluated by an experienced pathologist, based on defined characteristics whereby p16 was scored as positive if it was strong and diffuse (>70% of tumor cells), and negative if absent, weak, or focal [50 (link)].

The primary antibodies used for the immunohistochemical studies were the rabbit polyclonal anti-Vascular Endothelial Growth Factor Receptor 1 (VEGFR1) (ab32152, Abcam, United Kingdom) and anti-Vascular Endothelial Growth Factor Receptor 2 (VEGFR2) (ab2349, Abcam, United Kingdom). Eye sections of 5 μm thickness were deparaffinized in Bond Dewax solution (Leica Biosystems, Germany) and rehydrated prior to epitope retrieval in Novocastra Epitope Retrieval solution (Leica Biosystems, Germany) (just in case of VEGFR1). After 10 min incubation with 3% H2O2, followed by the blocking solution (Novocastra Leica Biosystems, Germany) also for 10 min, the tissue sections were incubated overnight at 4 °C with anti-VEGFR1 and anti-VEGFR2 antibodies (1:100 dilution). Detection was performed using a polymer detection system (RE7280-K, Novolink max Polymer detection system, Novocastra Leica Biosystems) and 3,3′- diaminobenzidine (DAB, Novocastra Leica Biosystems) as chromogenic substrate, according to the manufacturer’s instructions. Hematoxylin staining was applied before dehydration and mounting. Negative controls included substitution of the first antibody with normal rabbit serum. Images were acquired by light microscopy (Olympus BX43, Hamburg, Germany).

TMA blocks were sectioned at 2.5 μm. Sections were mounted on glass slides, dried and baked at 60°C for 30 min. H&E staining and double immunohistochemistry for Pan-CK and CD8 were performed. Double immunohistochemistry was performed using Bond RX (Leica Biosystems). Slides were dewaxed using Bond dewax solution (product code AR9222, Leica Biosystems). Heat-induced epitope retrieval in citrate buffer based (code AR9640, Leica Biosystems) at pH6 for 20 min at 100°C was followed by incubation with primary mouse pancytokeratin antibody (Dako, clone AE1/AE3, Ref M351501-2); dilution 1:400; for 30 min. Slides were incubated with HRP (horseradish peroxidase)-polymer for 15 min. Visualization was accomplished using 3,3-Diaminobenzidine (DAB) for 10 min, leading to a brown staining signal (Bond polymer refine detection, Leica Biosystems, Ref DS9800). As a second step, mouse CD8 antibody was used (Dako-Agilent, clone C8/144B, Ref M7103); dilution 1:100; incubation time 30 min. Alkaline Phosphatase (AP)-polymer was used as secondary antibody; incubation time 15 min. Visualization was accomplished using fast red resulting in a red chromogen (Red polymer refine Detection, Leica Biosystems, Ref DS9390). Samples were counterstained with hematoxylin and mounted with Aquatex (Merck).