Sc 1506

To prepare samples for histological analysis, the harvested organs were fixed in 10% formaldehyde in PBS (Rapid Fixative, Kojima Chemical Industry, Saitama, Japan) for 4 days at 4 °C, followed by dehydration, and, lastly, they were embedded in paraffin. The paraffin-embedded organs were sliced into 2–4 µm sections. Immunohistochemistry (IHC) staining for anti-CD 31 was carried out using an automated staining processor (Discovery XT, Ventana Medical Systems, Inc., Oro Valley, AZ, USA). The vascular endothelial cells’ IHC staining was carried out in pre-diluted polyclonal goat anti-CD31 antibody (1/100 dilution; sc-1506, Santa Cruz Biotechnology, Inc., Dallas, TX, USA; 2 h at room temperature) combined with a biotinylated anti-goat IgG (H + L) (BA5000, Vector Laboratories, Burlingame, CA, USA; 20 min at room temperature). Polyclonal rabbit anti-LYVE-1 antibody diluted in 1:250 (103-PA50AG, ReliaTech GmbH, Wolfenbüttel, Germany; 2 h at room temperature) combined with a biotinylated anti-rabbit IgG (H + L) (BA1000, Vector Laboratories, Burlingame, CA, USA; 20 min at room temperature) was used to carry out the IHC staining of vascular endothelial cells. A BX-51 Olympus microscope connected to a digital camera (DP72; Olympus corporation, Tokyo, Japan) was used to determine the specimen boundary at low magnification.

A standard Haematoxylin and Eosin (H&E) staining was performed to assess the morphology of tumours. Immunohistochemistry was carried out as previously described [27 (link)]. The following primary antibodies were used: mouse anti-MCT1 (1:500, sc-365501, Santa Cruz Biotechnology), rabbit anti-MCT4 (1:500, sc-50329, Santa Cruz Biotechnology), mouse anti-CD147 (1:400, sc-71038, Santa Cruz Biotechnology), as previously described [25 (link)], rabbit anti-CAIX (1:2000, ab15086, Abcam) and mouse anti-Ki67 (1:200, AP10243CM, Gennova), during 2 hours at room temperature, using UltraVision Detection System Anti-polyvalent, HRP (Labvision Corporation). Goat anti-PECAM (CD31, 1:400, sc-1506, Santa Cruz Biotechnology) diluted in PBS, was incubated overnight at room temperature, for further incubation with biotinylated horse anti-goat (1:500, BA-9500, Vector Laboratories) secondary antibody, 1 hour at room temperature. In the remaining steps, the R.T.U. Vectastain Elite ABC Kit (Vector Laboratories) was used. Antigen retrieval to Ki67 and CD31 reactions were performed with citrate buffer, in the microwave for 15 minutes.

Xenograft tumor and matrigel plug sections were deparaffinized with xylene and rehydrated with addition of ethanol. Heat-induced antigen retrieval was performed in Tris-EDTA buffer. Tissue sections were subjected to 3% hydrogen peroxide to remove endogenous peroxidase activity and then blocked with blocking buffer (P0102; Beyotime, China). Slides were incubated with primary antibodies against CD31 (sc-1506; Santa Cruz Biotechnology, Dallas, Texas, USA), VWF (sc-14014; Santa Cruz Biotechnology), CD34 (#3569; Cell Signaling Technology, MA, USA), and GFP (ARH2068; Antibody Revolution, San Diego, CA, USA) at 4°C overnight. Sections were washed three times for 5 minutes each time. For immunofluorescence analysis, fluorescein-labeled secondary antibodies (20014; 20106; Biotium, Hayward, CA, USA) were incubated at room temperature for 1 hour. Before immunofluorescence analysis with a confocal microscope, cells were counterstained with DAPI. For immunohistochemistry analysis, Real Envision Detection Kit (GK500710; Gene Tech, Shanghai, China) was used and signals were visualized through the diaminobenzidine reaction.

Transversal sections of 7 µm thick were obtained at cardiac midventricular level and stained using the Sirius Red/Fast Green kit (Chondrex). Fibrosis was assessed in all animals in four randomly chosen fields per section. The area of collagen deposition indicated by red staining was outlined and quantified using an automated image analysis program (Carl Zeiss, AxoVision 4.6, Zaventem, Belgium). Blood vessels were excluded. Total collagen deposition to the global cardiac area was calculated and expressed as percent collagen deposit.
Capillary density was quantified from histological sections by immunohistochemical staining for CD31 (SC-1506, Santa Cruz, 1/100). Capillaries were visualized by 3-3-diaminobezedine (DAB) and counterstained using hematoxylin. The amount of blood vessels were counted in 10 different fields per section and averaged. Data are expressed as amount of capillaries per µm².