Dmi8 sp8 microscope

Parental, Panx1-deleted and stable hPanx1 transfected N2a cells plated on glass bottom dishes were immunostained for Panx1. Cells fixed in 4% p-formaldehyde (PFA) were permeabilized with 0.2% Triton X-100 for 15 min, then incubated for 30 min in a blocking solution (2% BSA in PBS) and then overnight with rabbit anti-Panx1 antibody (1:200; Alomone, Jerusalem, Israel; cat#ACC-234) in blocking solution. After washout of the primary antibody, cells were exposed to donkey anti-rabbit secondary antibody (1:1000; Alexa Fluor 488, Invitrogen, Waltham, MA, USA; cat#A-21206) for 1 h at room temperature along with DAPI to stain nuclei, prior to mounting (ProLong Gold antifade, Invitrogen cat#P36930). Images were captured with an automated inverted Leica DMi8 SP8 microscope (Wetzlar, Germany) using a 63× NA = 1.4 Oil PLAPO, WD = 0.14 mm liquid immersion objective with a refractive index of 1.5. Leica LASX software (version number 3.3.0, Wetzlar, Germany) was used for image acquisition and analysis (Leica Microsystems CMS GmbH). Confocal images were collected using 488 nm laser beam and a pinhole diameter of 1 Airy unit.

Leica DMI8 SP8 microscope and LASX software was used for confocal imaging, live-cell imaging and image processing. For live-cell imaging, PLK4 overexpressing U2OS-H2B-mCherry cells were synchronized by double-thymidine treatment [53 (link)], metaphase cells were tracked and recorded based on the fate of division. Metaphase scoring was performed using Carl Zeiss Axio Imager M1. Competition assays were performed by using BioTek Cytation 5 Cell Imaging Multimode Reader.