Radiance 2000

Huh7 cells infected at greater than 90% were transfected with different plasmids as indicated in figure legends. At 24 h post-transfection, cells were trypsinized and grown on glass coverslips for another 24 h. The coverslips were then fixed with 4% formaldehyde in PBS for 10 min, washed in PBS and incubated in blocking buffer (PBS, 3% bovine serum albumin, 10% FBS, 0.1% Triton X-100) for 30 min at RT. For the detection of GFP-LC3, cells were permeabilized with 0.05% saponin to remove dispersed LC3 (LC3-I) [27 (link)]. After washing with PBS, the coverslips were incubated with primary antibodies in blocking buffer for 1 h at RT. Coverslips were then washed in PBS and incubated with either Alexa fluor-(488 or 568) goat anti-mouse IgG or Alexa fluor-(488 or 568) goat anti-rabbit IgG (Invitrogen) for 1h at RT. After washing, coverslips were mounted on glass slides with Prolong Antifade (Invitrogen) and examined with either a laser scanning confocal BioRad Radiance 2000 or a Zeiss LSM 780. The Manders’coefficient of colocalization was obtained using ImageJ software (NIH) in randomly selected regions that were positive for the targeted proteins from different cells. Manders’coefficient values over 0.4 were considered as strong colocalization.

Cells were fixed with 4% paraformaldehyde, blocked with 3% BSA, and incubated with anti-β-catenin antibody (1:100, BD Biosciences, #610153) overnight at 4 °C, followed by incubation with fluorescein isothiocyanate (FITC)-conjugated secondary antibodies, at room temperature for 1 h. Cells were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Confocal microscopy was performed using a Radiance 2000 laser scanning confocal microscope (Carl Zeiss, Thornwood, NY, USA).

Two-photon excitation by short infrared laser pulses was used to restrict emission collection to a thin focal excitation plane ~100 μm deep into the slice. We thus ensured that no contaminating fluorescence was collected from damaged tissue near the slice surface (no detectable autofluorescence from the slices was recorded before applying AF350). The imaging system was based on Biorad Radiance 2000 (Zeiss) microscope, integrated with a SPC-830 TCSPC Becker & Hickl imaging module and a SpectraPhysics MaiTai laser, pulsing at 80 Mhz with a pulse width of <200 fs and a wavelength of 790 nm optimized for Alexa Fluor 350 and Alexa Fluor 594 excitation22 (link)24 (link). Several objectives were used without digital zoom, 10× (NA 0.3), 20× (NA 0.5), 40× (water immersion NA 0.75), 63× (water immersion NA 1.2). Fluorescence was acquired at a laser line scanning rate of up to 500 Hz and routinely stored as a 256 × 256 × 256 (τ, x, y) data cube representing a stack of 8-bit x-y images. A short pass 700 nm filter was placed in front of the detector to block out any escaped light from the laser source. Average acquisition times varied between 30–300 s depending on the depth, and the maximum photon count rate was kept well at ~10⁵ s⁻¹ to avoid photon pile-up (maximal photon count of the system was near 10⁸ s⁻¹).