Tcs sp5 2 sp8

GIST-T1 and PC-9 cells were cultured on poly L-lysine-coated coverslips separately and fixed with 4% paraformaldehyde for 20 min at room temperature. The fixed cells were permeabilized and blocked for 30 min in Dulbecco’s phosphate-buffered saline (D-PBS(−)) supplemented with 0.1% saponin and 3% bovine serum albumin (BSA) and then incubated with primary and secondary antibodies for 1 h each. After washing with D-PBS(−), the cells were mounted with Fluoromount (Diagnostic BioSystems). Confocal images were obtained with a FLUOVIEW FV10i (Olympus, Tokyo, Japan) or TCS SP5 II/SP8 (Leica) laser scanning microscope. Composite figures were prepared using FLUOVIEW FV1000 Viewer (Olympus), Leica Application Suite X Software (Leica), Photoshop, and Illustrator software (Adobe).

Cells in suspension culture were fixed with methanol for 10 min at − 20 °C or with 4% paraformaldehyde for 20 min at room temperature, then cyto-centrifuged onto coverslips. GIST-T1 cells were cultured on poly-L-lysine-coated coverslips and fixed as above. Fixed cells were permeabilized and blocked for 30 min in PBS supplemented with 0.1% saponin and 3% BSA, and then incubated with a primary and a secondary antibody for 1 h each. After washing with PBS, cells were mounted with Fluoromount (DiagnosticBioSystems, Pleasanton, CA). Confocal images were obtained with an Fluoview FV10i (Olympus, Tokyo, Japan) or a TCS SP5 II/SP8 (Leica, Wetzlar, Germany) laser scanning microscope. Composite figures were prepared with Photoshop Elements 10 and Illustrator CS6 software (Adobe, San Jose, CA). Pearson’s R were calculated with NIH ImageJ 1.48v software.

Leukemia cells in suspension culture were fixed with 4% paraformaldehyde (PFA) for 20 min at room temperature, then cyto-centrifuged onto coverslips. Fixed cells were permeabilized and blocked for 30 min in phosphate-buffered saline (PBS) supplemented with 0.1% saponin and 3% bovine serum albumin (BSA), and then incubated with a primary and a secondary antibody for 1 h each. AF647-conjugated lectin-Helix pomatia agglutinin (lectin-HPA, Thermo Fisher Scientific) was used for Golgi staining. After washing with PBS, cells were mounted with Fluoromount (DiagnosticBioSystems, Pleasanton, CA). For staining the extracellular domain of FLT3, living MOLM-14 cells were stained with anti-FLT3 (SF1.340) and AF488-conjugated anti-mouse IgG in PBS supplemented with 3% BSA and 0.1% sodium azide (NaN₃) at 4 °C for 1 h each. Stained cells were fixed with 4% PFA for 20 min at room temperature. Confocal images were obtained with an Fluoview FV10i (Olympus, Tokyo, Japan) or a TCS SP5 II/SP8 (Leica, Wetzlar, Germany) laser scanning microscope. Composite figures were prepared with an FV1000 Viewer (Olympus), LAS X (Leica), Photoshop, and Illustrator software (Adobe, San Jose, CA).