A1r inverted laser scanning confocal microscope

Keratinocytes with or without 2mM CaCl₂ (denoted as Ca²⁺) or MgCl₂(denoted as Mg²⁺) as indicated were fixed after no treatment or treated with DMSO, BTT3033 (20μM), ML141 (10μM). The cells were fixed with 4% paraformaldehyde (PFA) for 10 min and permeabilized with 0.1% Triton X-100. Cells were incubated with primary antibodies for 2 h and with appropriate secondary antibodies conjugated to Alexa Fluor 568 or 488 (1:1000) and phalloidin conjugated to Alexa Fluor 647 (1:500), including Hoechst, for 1 h. Cells were mounted on slides using FluorSave (ICN). Normal human skin sections were processed for staining as previously described [61 (link)]. Confocal microscopy was performed using a Nikon A1R inverted confocal laser scanning microscope with a 60x oil objective and laser excitation wavelengths of 405, 488, 561 and 633 nm. Some samples were imaged using a structured illumination microscopy (N-SIM, Nikon) using an iXon3 EM-CCD camera (Andor), respectively. Images within the same experiments were all acquired at the same laser settings using Nikon NIS Elements software.

MEFs were seeded at low confluency in imaging chambers (ibidi, 80826) and transfected with mRFP-Lifeact and EGFP-FSCN-1 at 24 h post seeding. Bbs6 wild-type and knockout MEFs were imaged at 48 h after transfection with a Leica SP8 confocal microscope with photomultipliers and a HyD detector (HC PL APO 63×/1.40 OIL, Leica) under the conditions of 37°C supplied with 5% CO₂. Videos of Bbs8 wild-type and knockout MEFs in an environmental chamber maintained at 37°C/5% CO₂ were acquired on a Nikon A1R inverted laser scanning confocal microscope (Nikon Instruments) combined with a 60× Plan Fluore oil immersion objective (NA 1). Excitation wavelengths of 488 nm and 561 nm (diode lasers) were used. To follow their movement, cells co-expressing mRFP-Lifeact and EGFP-FSCN-1 were imaged every 5 sec for 30 timepoints. Videos were acquired either with the Leica LAS X (version 3.5.7.23225) or Nikon NIS-Elements (v4) imaging software.