Ab53066

For IF staining, cells at 1 × 10⁴ were seeded on sterile glass coverslips for 24 h, fixed in cold absolute methanol, and subjected to staining with antibodies against epithelial cytokeratin (CK)‑19 and pan CK. Specific markers for stromal fibroblasts—α‑SMA and FAP—were used for quality control of the cancer cell purity. CSF1R was evaluated in the obtained TGCT cells. Briefly, cells were permeabilized with 0.2% Triton‑1X PBS and incubated overnight at 4 °C with the following primary antibodies: mouse anti‑human panCK antibody (sc‑8018; Santa Cruz Biotechnology Inc.); mouse anti‑human CK‑19 antibody (Santa Cruz Biotechnology Inc.); mouse anti‑human α‑SMA antibody (Sigma‑Aldrich; Merck KGaA); rabbit anti‑human fibroblast activation protein (FAP) antibody (ab53066; Abcam); and rabbit anti‑human CSF-1R antibody (ab205921; Abcam). The goat anti‑mouse IgG‑Cy3 antibody (#115‑166‑071; Jackson ImmunoResearch Laboratories Inc.) or the donkey anti‑rabbit IgG (H + L) highly cross‑adsorbed secondary antibody Alexa Fluor 488 (21,206; Thermo Fisher Scientific Inc.) was used. The nuclei were stained with Hoechst 33,342 (Invitrogen; Thermo Fisher Scientific Inc.). Fluorescence was captured with a ZEISS LSM 800 confocal laser fluorescence scanning microscope (Axio Observer 7 LSM 800; Zeiss GmbH).

For ICC, cells were seeded on a glass coverslip and cultured with control medium or CM from MIApaca‐2, BxPC‐3, SW1990 or PANC‐1 for 24 h. Cells were washed and fixed. Triton‐X‐100 was used to permeabilize the cells. After treating with 3% hydrogen peroxide, cells were incubated with antibodies against α‐SMA (ab32575, 1:100) and PDGFR‐α (ab203491, 1:100) from Abcam overnight, and horseradish peroxidase‐linked secondary antibodies for 1 h. Diaminobenzidine (DAB) was used for detection. For IF, cells were treated as in ICC and incubated with antibodies against α‐SMA (ab32575, 1:100), E‐cadherin (ab219332, 1:100), fibroblast activation protein (FAP, ab53066, 1:100) and epithelial cell adhesion molecule (EpCAM) (ab218448, 1:100) from Abcam overnight. The cells were then incubated with the secondary antibodies, Alexa Fluor 488 (A32731, Invitrogen) and Alexa Fluor 594 (A48271, Invitrogen). Subsequently, the coverslips were fixed and imaged.