Fluoview fv10i laser scanning microscope

The following antibodies were purchased: anti-KIT (M-14) from Santa Cruz Biotechnology (Dallas, TX); anti-KIT (D13A2) and anti-phospho-KIT (Y703) from Cell Signaling Technology (Danvers, MA); anti-GM130^{34 (link)} from BD Transduction Laboratories (Franklin Lakes, NJ) and anti-PDI from Abcam (Cambridge, UK). Alexa Fluor-conjugated secondary antibodies were obtained from Molecular Probes (Eugene, OR).
Cells cultured on poly-L-lysine-coated coverslips were fixed with 4% paraformaldehyde for 20 min at 25 °C. The fixed cells were permeabilised and blocked for 30 min in PBS supplemented with 0.1% saponin and 3% BSA, and then incubated with a primary and secondary antibody for 1 h each. Confocal images were obtained using a Fluoview FV10i laser scanning microscope with an x60 1.20 N.A. water-immersion objective (Olympus, Tokyo, Japan). Composite figures were prepared with Photoshop Elements 10 and Illustrator CS6 software (Adobe, San Jose, CA).

Cells were fixed with 4% PFA for 20 min at room temperature, or with methanol for 10 min at −20 °C, then cyto-centrifuged onto coverslips. Fixed cells were permeabilized and blocked for 30 min in PBS supplemented with 0.1% saponin and 3% bovine serum albumin, and then incubated with a primary and a secondary antibody for 1 h each. To stain with anti-Akt(pSer473) (Cell Signaling Technology; 193H12), 10% skimmed milk was used for blocking. After washing with PBS, cells were mounted with Fluomount (DiagnosticBioSystems). For staining endocytic compartments, cells were incubated for 1 h with 5 μg ml⁻¹ AF647-CTXB or 1 mg ml⁻¹ AF647-dextran (Molecular Probes). Confocal images were obtained by a Fluoview FV10i laser scanning microscope with an x60 1.20 N.A. water-immersion objective (Olympus). Composite figures were prepared with Photoshop elements 10 and Illustrator CS6 software (Adobe). Pearson’s correlation coefficients (Pearson’s R) were calculated with NIH ImageJ Version 1.48v software.

Cells were fixed with methanol for 10 minutes at -20°C, then cyto-centrifuged onto coverslips. Fixed cells were permeabilized and blocked for 30 minutes in PBS supplemented with 0.1% saponin and 3% BSA, and then incubated with a primary and secondary antibody for 1 hour each. After washing with PBS, cells were mounted with Fluoromount (DiagnosticBioSystems, Pleasanton, CA). Confocal images were obtained with a Fluoview FV10i laser scanning microscope with an x60 1.20 N.A. water-immersion objective (Olympus, Tokyo, Japan). Composite figures were prepared with Photoshop Elements 10 and Illustrator CS6 software (Adobe, San Jose, CA). Pearson’s R correlation coefficients were calculated with NIH ImageJ 1.48v software.