Plan apochromat 40 1.3 oil dic m27

The culture inserts were fixed in 10% buffered formaldehyde, permeabilized with 0.1% Triton X-100, and blocked with 1% BSA in PBS-Tween 20^TM for 30 min under rotation (room temperature). A keratin 1 (CK1) primary antibody was used for detection and a secondary Alexa 488-conjugated antibody was used for visualization (Finetest, Hubei, China). Hoechst 33342 (Thermo Fisher Scientific, USA) was used for nuclei visualization. Images were obtained using an inverted confocal laser scanning microscope Zeiss LSM 880 with AiryScan, a 32-channel GaAsP-PMT area detector, a Plan-Apochromat 40×/1.3 Oil DIC M27 (WD = 0.2 mm), and a (UV) VIS-IR objective with Scan zoom 2. A Z-stack of a 27–30 µm depth was scanned for one field of view in two randomly chosen locations for each sample with a 0.1 µm × 0.1 µm pixel size and a 106.27 µm × 106.27 µm image size in AiryScan SR mode with a 2.05 µs pixel time. AiryScan processing, Z-stack maximal intensity projection, and histogram Min/Max correction were performed using the Zeiss ZEN 2.3. Zeiss LSM 880 with Airyscan is equipment belonging to the Core Centrum of the Institute of Developmental Biology RAS, Moscow, Russia.

FRAP experiments were performed with a confocal laser-scanning microscope ZEISS LSM 880 (Carl Zeiss AG, Oberkochen, Germany) equipped with an Airyscan detection unit and a high sensitivity GAsP detector for visible detection. To maximize the resolution enhancement, we used a high numerical aperture (NA) oil immersion objective (Plan-Apochromat 40×/1.3 Oil DIC M27; Zeiss). Two spectral channels were used to record the fluorescence of NBD-PC lipid (laser excitation: 458 nm; emission: 470–600 nm) and A5-Alexa 647 (laser excitation: 633 nm; emission: 640–750 nm). The photobleaching was achieved using a 405 -nm laser operated at maximum laser power. FRAP experiments were successfully performed in replicates for more than three times on different samples and days.