Aec101 1kt

Detection of IDO, PD-L1 and IL-27 by immunohistochemistry (IHC) was performed on sections of formaldehyde-fixed paraffin-embedded tumors explanted from mice or from 6 high grade serous EOC. Antigen-retrieval was performed with pH 6.0 10 mM citrate buffer (for PD-L1 and IL-27) or 1 mM EDTA, 0.05% tween20 pH 8.0 buffer (for IDO) in microwave oven. The sections were stained using rabbit anti-IL-27A antibody (LifeSpan BioSciences, LS-B2719-LSBio), anti-IDO mAb (Chemicon, clone 10.1, MAB5412) or rabbit anti-PD-L1 antibody (ProSci, 4059) overnight at 4°C. The antibody complex was revealed with the anti-rabbit or anti-mouse EnVision+ System-Peroxidase (Dako, K4002 and K4000, respectively) and 3-amino-9-ethylcarbazole (Sigma-Aldrich, AEC101-1KT). The sections were counterstained with modified Mayer's ematoxylin solution (Sigma-Aldrich, MHS16) and mounted in Fluoromount Aqueous Mounting Medium (Sigma-Aldrich, F4680). Images were captured with a Nikon Eclipse 80i light microscope equipped with a color camera, using a 40x objective.

Tissues were dissected out; fixed in Bouin’s fixative overnight, cryo-protected and serial sections were cut on a cryostat (CM1850; Leica) at 4–5 μm thickness. The tissue sections were washed with PBS, antigen retrieval was done with enzymatic retrieval method using trypsin-CaCl₂ solution. Sections were blocked with 1% BSA at 37 °C. Sections were incubated overnight at 25 °C in a humid atmosphere with primary antibodies against CD31 (1:100; Santa Cruz Biotechnology, Inc.). The sections were and then incubated with biotinylated anti-mouse IgG followed by peroxidase conjugate extra-avidin (Sigma-Aldrich; 1:100) for 60 minutes and 3,3′-diaminobenzidine was used as chromogen (Sigma-Aldrich AEC101-1KT; 1:100) to visualize the reaction product and counterstained with hematoxylin (1:1; Himedia, India). Finally, sections were washed in distilled water and mounted in glycerol gelatin. Kidney and liver sections were cut on a cryostat at 4–5 μm thickness. Sections were stained with haematoxylin and eosin. Images were acquired with a bright-field microscope (Leica) at 10x magnification.