Mouse anti human ck 19 antibody

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).

To identify primary cultures of fibroblasts from contaminating cancer cells, the absence of epithelial markers and the presence of fibroblast-related mesenchymal markers were checked. Cytokeratin 19 (CK19), an epithelial marker; vimentin (VIM) and alpha-smooth muscle actin (ASMA), mesenchymal markers, were selected. CCA cell lines were used as the positive controls for CK19 detection. Cells were cultured for 24 h in 96-well plates and fixed with 4% paraformaldehyde for 15 min and permeabilized with 1% Triton X-100 for 1 min. Non-specific binding was blocked by 1% BSA in 1X PBS for ASMA and 5% FBS in 1X PBS for VIM and CK19. The mouse anti-human CK19 antibody (1:100 dilution; SC-6278, Santa Cruz Biotechnologies Inc.), mouse anti-human VIM antibody (1:500 dilution; SC-6260, Santa Cruz Biotechnologies Inc.), and mouse anti-human ASMA antibody (1:200 dilution; Sigma-Aldrich) were used. The primary antibody was incubated for 3 h at room temperature. After washing, goat anti-mouse IgG-Cy3 (Jackson Immunoresearch Laboratories Inc.) was added for 1 h at room temperature followed by adding Hoechst stain (Invitrogen). The signals were detected under an inverted fluorescence microscope.