Jc70a

Our experiments were performed on two samples of human granulation tissue. The first sample was a set of eight consecutively sliced tissue sections of H&E-stained granulation tissue. The second one was a set of ten consecutive tissue sections of a CD31-stained granulation tissue. The thickness of the sections was 4 µm for the H&E and 3 µm for the CD31 samples. For both staining methods, the fresh tissue was first fixed in 5% formalin and then embedded in paraffin. The formalin-fixed paraffin-embedded (FFPE) tissue blocks were cut into consecutive sections and mounted onto glass slides. For the H&E staining, the tissue was deparaffinized, rehydrated and then stained in Mayer’s hematoxylin and eosin. The immunohistochemical staining of CD31 was performed with the help of a BenchMark Ultra autostainer (Ventana Medical Systems, Oro Valley, AZ, USA) employing a mouse anti-CD31 antibody (1:40; clone JC70A; Dako, Glostrup, Denmark). After staining both the H&E and CD31, the slides were covered with glass cover slips and were scanned as whole slide images (WSI) via the PreciPoint M8 slide scanner (PreciPoint GmbH, Freising, Germany) with a 20× objective (Olympus UPlan FLN 20×, Olympus, Tokyo, Japan). The resolution observed at maximum zoom was 0.28 µm per pixel.

Formalin-fixed, paraffin-embedded specimens from a total of 30 primary ccRCCs (Supplementary Table 1) were retrieved from the archives of the Department of Pathology of the University of Heidelberg School of Medicine under Ethics committee vota 206/2005 and 207/2005, and informed consent by the patients. Sections were deparaffinized in xylene and rehydrated in a graded ethanol series. Antigen retrieval was performed with a steam cooker using retrieval buffer (Target Retrieval Solution, Dako). Primary antibodies used were as follows: HIF-1α (Novus Biologicals, H1alpha67, NB100-105, 1:100), HIF-2α (Novus Biologicals, NB100-122, 1:100), phospho-mTOR S2448 (Cell Signaling, 49F9, #2976, 1:100), phospho-S6RP S235/236 (Cell Signaling, #2211, 1:50), Ki-67 (Dako, MIB-1, M7240, 1:100), CD31 (Dako, JC70A, M0823, 1:100) and CD45 (Dako, 2B11+PD7/26, M0701, 1:100). Immunodetection was performed using the Histostain-Plus Detection Kit (Invitrogen) according to the manufacturer's recommendations. The immunostaining for HIF-1α has been validated in a previous study using a ccRCC with a deletion in exon 2 of the VHL gene32 (link).

For immunofluorescent staining, 7 μm acetone-fixed cryosections were thawed and then blocked for non-specific binding by incubation in PBS with 10% goat serum and casein solution, for 30 min at RT. This was followed by 1 h incubation with primary antibodies for SCARF-1 (8 μg/ml, Abcam ab92308) and CD31 (5 μg/ml, DAKO JC70A). Samples were washed three times in PBS followed by 30 min incubation with Alexa Fluor® conjugated secondary antibodies (1:500 dilution; Thermo Fisher Scientific). Nuclei were stained with 300 nM DAPI (Invitrogen) and slides were subsequently mounted with ProLong™ Gold Antifade Mountant (Invitrogen). Fluorescence images were acquired using a Zeiss 780 Zen confocal fluorescence microscope (ZEISS).

Immunohistochemistry was performed on a Leica Bond system using the standard protocol F30. The sections were pre-treated using heat mediated antigen retrieval with citrate-based buffer (pH 6, epitope retrieval solution 1) or EDTA based buffer (pH 9, epitope retrieval solution 2) for 20 min. The sections were then incubated with antibody for 30 min at room temperature and detected using an HRP conjugated polymer system in which DAB was used as the chromogen. The sections were counter-stained with haematoxylin and mounted with Aquatex. The following antibodies were used; anti-Pentraxin 3/PTX3 1/500 [MNB1] (Abcam 90806), anti-Cytokeratin 7 1/400 [RCK105] (Abcam 9021), anti-ITGA7 1/500 (Abcam 203254), anti-MGP 1/500 (Abcam 86233), anti-TEM1 1/400 (Abcam 67273), anti-CD31 1/800 [JC70A] (DAKO 20057487), anti-CD68 1/400 (DAKO 20058607), anti-Smooth muscle actin 1/1000 (Abcam 5694), anti-periostin 1/1500 [EPR20806] (Abcam 227049), anti-APOD 1/500 (orb155698), anti-CXCL14 1/1200 (Abcam 137541), anti-Podoplanin 1/750 [D2-40] GTX31231 (lot 821903108).

For immunofluorescence, 4 μm paraffin sections were deparaffinized, and tissue slides were steam heated for 30 minutes at 95°C (citrate buffer pH 6.1) for antigen retrieval followed by a 10% BSA blocking step. Sections were incubated over night at 4°C with a CD13 antibody (SP187, Cell Marque; dilution 1:300) and a CD31 antibody (JC70A, DAKO, dilution 1:200) followed by a 60 min incubation with a Cy3-labelled goat anti-rabbit-IgG antibody (#111-165-003, Dianova; dilution 1:200) and a FITC-labelled goat anti-mouse-IgG (#554001, BD Pharmingen; dilution 1:500). Appropriate PBS washing steps were employed, nuclei were counterstained with DAPI using a standard procedure. Since the antibodies used for CD13 and CD31 staining are species-specific for human CD13 and CD31, vascular and perivascular staining were not assayed in the xenografts.

Immunohistochemistry was used to detect multiple inflammatory tissue markers in cryosections. Tissue was left to dry overnight and the following day it was fixed in acetone at 4 °C for 10 min and washed in PBS 2 × 5 min at room temperature. Sections were blocked in 10% goat serum for 1 h and H₂O₂ for 5 min. Each blocking step was followed by washing in PBS for 2 × 5 min. Carotids were then incubated in primary antibody for 30 min at room temperature. Antibodies included CD68 (1:500, clone PG-M1), CD31 (1:20, clone JC70A) and Smooth Muscle Actin (1:500, clone 1A4) (all from Dako UK, Ely, UK). Labelled polymer-HRP anti-rabbit application and visualization with 3,3′-diaminobenzidine (DAB) was done using EnVision+ System-HRP (DAB) kit (Dako UK, Ely, UK), following manufacturers specifications. Counterstain of 150 μl haemotoxylin was applied to each section and incubated for 1 min. Slide was then dipped six times in destain solution (1% HCl, 50% methanol, 49% distilled H₂O) and washed in tap water for 2 min. Afterwards tissue was quickly dehydrated in 1 min intervals through graded alcohols (30 to 70% to 100% to absolute alcohol 1 to absolute alcohol 2) and cleared in xylene for at least 1 h.