Ab93283

For in vivo studies, paraffin-embedded heart tissue sections were deparaffinized and rehydrated, blocked in 10% bovine serum albumin (BSA) (Thermo Fisher Scientific, MA, USA), and incubated with primary antibodies, specific anti-GFP antibody (1:100 dilution; ABclonal, AE011), anti-FSP1 antibody (1:50 dilution; Abcam, ab93283), anti-Snail1 antibody (1:100 dilution; eBioscience, 14-9859-82), and anti-MCT1 antibody (1:100 dilution; ABclonal, A9061) at 4°C overnight. For in vitro studies, endothelial cells were fixed with 3.7% formaldehyde (Sigma-Aldrich, MO, USA), permeabilized with 0.1% Triton X-100 (Sigma-Aldrich, MO, USA) in phosphate-buffered saline (PBS), and blocked with 3% BSA in PBS followed by incubating with primary antibodies at 4°C overnight. The following primary antibodies were used for immunofluorescent staining: anti–VE-cadherin antibody (1:50 dilution; Abcam, ab33168), anti-CD31 antibody (1:50 dilution; Abcam, ab28364), anti-FSP1 antibody (1:50 dilution; Abcam, ab93283), and anti-Snail1 antibody (1:100 dilution; eBioscience, 14-9859-82). The next day, slides or cells were stained with secondary antibodies at room temperature for 90 min and mounted in mounting medium with DAPI (Vector Laboratories, CA, USA).

sEV protein content was measured by BCA, and 10 μg aliquots were used for Western blot analysis. sEVs were lysed in Laemmli buffer at 96°C for 5 minutes to denature proteins, and protein extracts were resolved by SDS-PAGE. Antibodies were tested against albumin (sc-271605, Santa Cruz Biotechnology), apolipoprotein B (ab139401, Abcam), TSG101 (ab125011, Abcam), CD9 (ab58989, Abcam), and ALIX (ab88743, Abcam). Antibodies were chosen according to MISEV 2023 guidelines (21 (link)). Immunostaining against PAX8 (PA 0300, Biopat), a well-known ovarian carcinoma marker, was also incorporated in the Western blot characterization panel. The biological validation used antibodies against STX5 (HPA001358, Sigma-Aldrich) and S100A4 (ab93283, Abcam). Clarity ECL Western Blotting Substrate (Bio-Rad) was used to visualize the bands and developed on the ChemiDoc imaging system (Bio-Rad). The intensity of the immunoreactive bands was quantified by densitometry using ImageJ (NIH).

Formalin-fixed and paraffin-embedded tumor specimens of 14 CCA patients were analyzed for the presence of CAF and SC and correlated with survival rates. All patients signed the informed consent. The study was approved by the local institutional review board of the Medical Faculty of the Kiel University (A 110/99). For immunohistochemistry, 5 µm paraffin sections were used. Antigen retrieval was not necessary. Slides were incubated with primary antibodies for α-SMA (1:400, clone 1A4, mouse, NeoMarkers, Fremont, CA, USA), FSP-1 (1:200, abcam ab93283, Cambridge, UK) and S100 (1:400, Z0311, polyclonal, rabbit, Dako, Glostrup, Denmark). Bound antibodies were detected by EnVision+System-HRP anti-mouse antibody (Dako, Glostrup, Denmark). Color development was performed with the DAB substrate kit (Dako, Glostrup, Denmark). All slides were counterstained with hemalum and cover slipped. Evaluation of the stainings was performed on a Leica DM 1000 microscope (Leica, Wetzlar, Germany). The intensity of the staining was judged on an arbitrary scale of 0 to 3 with 0: no staining; 1: weak staining; 2: moderate staining and 3: strong staining by two independent pathologists.