Tcs sp8 smd microscope

H69 cholangiocytes were seeded in 8-well chambered coverglass and incubated for 6 days in H69 culture medium without fetal bovine serum that was supplemented with 20% serum of patients with PSC or IRC. Medium including patient serum was refreshed daily. Serum treatment was carried out in a blinded fashion. At day 4 after seeding, cells were transiently transfected with ANO1-mCherry. At day 6 after seeding, H69 medium was changed for Leibowitz imaging medium without phenol red (Thermo Scientific, Waltham, MS) supplemented with 20% of the respective patient serum. In addition, CellMask Green (Thermo Scientific, Waltham, MS) was added to the imaging medium to stain the plasma membrane. Microscopy was performed using a Leica TCS SP8-SMD microscope (LEICA, Wetzlar, Germany). Semiquantitative scoring of ANO1-mCherry plasma membrane staining was done separately in a blinded fashion by 3 researchers. Per ANO1-mCherry transfected cell, the ANO1-mCherry localization pattern was categorized as ‘strong membrane localization’, ‘light membrane localization’, or ‘no membrane localization’.

Confocal images and 3D stacks were acquired using a Leica TCS SP8 SMD microscope equipped with a 40x HC PL APO oil objective. Pinhole size was adjusted to 1.0 airy units and sequential scanning was performed at 400 Hz. 405nm, 488nm and 561nm laser lines were used for excitation.