Tsc spe confocal microscope

The generation of intracellular ROS was detected using the CellROX Deep Red fluorescent probe (Invitrogen). The cells were treated with IC₉₀ of STS for 24 h and exposed to CellROX Deep Red (5 μM, 30 min) at 26 °C in the dark. Cells were observed in a Leica TSC SPE- confocal microscope equipped with inverted optics at λ_exc = 633 and λ_em = 519 nm.

The generation of intracellular ROS was detected using the CellROX® Deep Red fluorescent probe (Invitrogen). The cells were treated with IC90 of both 5 and 7 for 24 h and exposed to CellROX® Deep Red (5 μM, 30 min) at 26 °C in the dark. Cells were observed in a Leica TSC SPE-confocal microscope equipped with inverted optics at λexc = 633 and λem = 519 nm.

Fixed samples were imaged using an inverted Leica TSC SPE confocal microscope. For representative images, a 60×/1.40NA oil immersion objective was used. For 4X scans a z-step size of 0.3 μm was used.
Live samples were imaged with an Andor revolution spinning disc confocal system, consisting of a Yokogawa CSU-X1 spinning disk unit and two Andor iXon3 DU-897-BV EMCCD cameras. A 60×/1.4NA oil immersion objective mounted on a Nikon Eclipse Ti microscope was used. Live imaging voxels sizes are 0.22 × 0.22 × 0.5 µm (60x/1.4NA spinning disc).

PLC3^WT, PLC3^SHARPIN-KO and PLC3^RNF5-KO were seeded in black 96-well plates and exposed to a dilution of HEV preparation with an MOI of 22,000, defined as 1 IU/cell, and IU (International unit) was quantified by qPCR as described previously [23 (link)]. To determine the infectivity titer, cells were fixed by methanol, followed by incubation with 0.5% Triton X-100 and blocking with PBS containing 10% goat serum. HEV ORF2 staining was performed using a mouse ORF2-specific monoclonal antibody (1E6, Merck, Darmstadt, Germany) in combination with an Alexa Fluor 488-conjugated goat anti-mouse antibody. Nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole). Microscopy was performed using a Leica TSC-SPE confocal microscope (10× or 20× objective) (Wetzlar, Germany). Infectivity of the respective well was calculated as a percentage with automated cell counting as well as ORF2 fluorescence with ImageJ software (v1.54d).

EAhy cells were seeded on glass slides, 24 hours later, they were treated with either scramble siRNA or siRNA against ShcA. 48 hours post-transfection, the cells were fixed with paraformaldehyde, and incubated with anti-ZEB1 or anti-IgG control primary antibodies and Alexa Fluor 488 secondary antibodies. Immunofluorescence-labeled cells were analyzed using a Leica TSC SPE confocal microscope with the ×63 oil immersion objective.

The SYTOX Green assay was performed to detect alterations of the membrane permeability in treated cells. Briefly, 10⁵ trophozoites were washed and incubated in saline solution with the SYTOX Green at a final concentration of 1 μM (Molecular Probes) for 15 min in the dark. Subsequently, DT solution was added (IC₉₀). After 24 h of treatment, cells were observed in a Leica TSC SPE confocal microscope equipped with inverted optics at λ_exc 482 nm and λ_em 519 nm.

Immunostaining for amylase, LAMP1, LAMP2, TFEB, and V-ATP6V1A was performed on pancreatic tissue cryosections. Images were acquired using a Leica TSC SPE confocal microscope with a 63× objective (Leica, Mannheim, Germany). Nuclei were counterstained with Hoechst33342 (H3570; Thermo Fisher Scientific). To assess the intensities of colocalization of 2 proteins, ImageJ 1.47v (National Institutes of Health, Bethesda, MD) colocalization plug-in was used (https://imagej.nih.gov/ij/plugins/colocalization.html).