Mouse anti tsg101

To analyze the intracellular localization of CT, Me665cells (4 × 10⁴) were cultured on 24-well plates containing coverslips until 60–70% of confluency. Cells were incubated at 37 °C with or without 7 µM BODIPY C16 for 4 h. 12 nM CT was added for 20 min on ice (T0), and cells were then incubated at 37 °C for 24 h (T24). Cells were fixed with 3 % paraformaldehyde (PFA) for 20 min at RT and permeabilized with 0.05% saponin. The primary antibodies used were: rabbit polyclonal anti-Cholera toxin (Sigma-Aldrich), mouse anti-TSG101 (GeneTex, Hsinchu, Taiwan), and mouse anti-LBPA/BMP (6C4) (Echelon Corporation, San Jose, CA, USA). AlexaFluor647-conjugated and AlexaFluor488-conjugated goat anti mouse or anti rabbit (Life Technologies) was used as secondary antibody. Coverslips were mounted on the microscope slide with Vectashield antifade mounting medium containing DAPI (Vector Laboratories, Burlingame, CA, USA). Images were taken by a FV1000 confocal microscope (Olympus, Tokyo, Japan), using a (Olympus) planapo objective 60x oil A.N. 1.42. Excitation light was obtained by a Laser Dapi 408 nm for DAPI, an Argon Ion Laser (488 nm) for FITC (Alexa 488), and a Red Diode Laser (638 nm) for Alexa 647. DAPI emission was recorded from 415 to 485 nm, FITC emission was recorded from 495 to 550 nm, and Alexa 647 from 634 to 750 nm. Images recorded have an optical thickness of 0.3 mm.