Hc pl apo cs2 1.4 na oil objective

The Leica TCS SP8 confocal microscope was equipped with an environment-controlled chamber (LIFE IMAGING SERVICES, Switzerland) maintained at 37°C and a HC PL APO CS2 1.4 NA oil objective (Leica Microsystems, Wetzlar, Germany). Alexa488-conjugated secondary antibodies, fluorescent fusion proteins containing mCitrine and mCherry, and EGF-Alexa647 were excited using a 470-670 nm white light laser (white light laser Kit WLL2, NKT Photonics, Denmark) at 488, 514 and 561 and 647 nm, respectively. PH-Akt-Cerulean was excited using an Argon Laser at 458 nm. Detection of fluorescence emission was restricted with an AOBS as follows: Cerulean @ 468-505 nm, Alexa488 @ 498-551 nm, mCitrine @ 525-570 nm, mCherry @ 570-650 nm and Alexa647 @ 654-754 nm. The pinhole was set to 250 μm and 12-bit images of 512x512 pixels were acquired in a frame-by-frame sequential mode.