Prolong glass antifade reagent

Caco-2bbe/NHE3 cells grown on Transwells (Corning, Lowell, MA) were fixed using 100% methanol at −20°C for 20 min. Cells were washed three times with PBS, permeabilized using 0.05% Triton X-100 in PBS, and blocked with 5% normal goat serum for 1 h at RT. Cells were then stained with mouse anti-VSVG P5D4 antibody for 16 h at 4°C. Cells were washed with PBS three times and incubated with Alexa 488-conjugated goat anti-mouse IgG (Invitrogen) and Alexa Fluor 647 phalloidin (ThermoFischer) for 1 h at RT. After three 10 min washes with PBS, specimens were mounted with ProLong Glass Antifade Reagent (Invitrogen) and observed under a Nikon A1R HD confocal microscope (Nikon Instruments Inc., NY) coupled to a Plan Apo λ 60x Oli lens.

48 hours after transfection, cells were fixed with 4% paraformaldehyde/PBS solution for 10 min. at room temperature, washed with PBS, and permeabilized in 0.1% TritonX-100/PBS for 1 min. Cells were incubated with 1 μM of SNAP Surface Alexa Fluor 488 (New England BioLabs #S9129S, Ipswich, MA) and 1:1000 ER Staining Kit-Red Fluorescence-Cytopainter (Abcam #139482, Cambridge, MA) at 37 °C for 30 min. protected from light. Hoechst 33342 was used for nuclear staining. Cover slips were mounted using ProLong Glass antifade reagent (Thermo Fisher #P36982). Confocal fluorescence microscopy was performed using Leica SP8X laser scanning confocal microscope equipped with a 40x oil immersion objective (Leica Camera, Wetzlar, Germany). The detection pinhole was set to 1 Airy unit, light collection configuration was optimized according to the combination of chosen fluorochromes (Alexa Fluor 488, Texas Red, and Hoechst), and sequential channel acquisition was performed to minimize the risk of bleed-through. The intensity gain was adjusted for each channel before capture in order to avoid saturated pixels. 8 bit, 1024 × 1024 pixel images were collected as Z-stack acquisition. All microscopy was performed in collaboration with the W.M. Keck Microscopy center on the University of Washington School of Medicine campus.

Huh7 cells were seeded on glass coverslips in 24-well plates at a density of 20,000 cells per well and grown overnight. The next day, cells were infected with ZIKV at a MOI of 10 and/or transfected as needed, and grown another 24 h before fixation with 4% paraformaldehyde in PBS for 15 min at room temperature. After washing with PBS, cells were blocked/permeabilized in blocking buffer (10% normal goat serum, 0.1% Triton X-100 in PBS) for 1 h. Incubations with primary and Alexa Fluor-conjugated secondary antibodies diluted in blocking buffer (see “Reporting summary” for a list of antibodies and dilution information) were performed for 1 h at room temperature, separated by three 5-min washes with PBS. DAPI (4′,6-diamidino-2-phenylindole) was used to visualize nuclei. Coverslips were mounted on glass slides with ProLong Glass antifade reagent (Thermo Fisher). Images were acquired with a Zeiss LSM 880 laser-scanning confocal microscope in Airyscan mode using a 63×/1.4 NA oil objective. Fluorophores were excited sequentially by 405-/488-/561-/633-nm lines, and imaging conditions were optimized to minimize bleed-through. Z stacks were taken with a 0.18-μm interval between slices. Airyscan processing was performed in Zeiss Zen software using the default settings.

Cells were grown on coverslips, fixed with 4% PFA for 15 min, permeabilized with 0.1% Triton X‐100 for 10 min and blocked with 1% BSA in PBS for 30 min at RT. Cells were then stained with primary antibody for 1 h at RT followed by secondary antibody for 1 h at RT. Cover slips were rinsed in H₂O and mounted on slides using Prolong glass antifade reagent (Thermo Fisher Scientific). For detection of internalized integrin β1, cells were incubated with 2 μg/ml integrin β1 at 37°C for 30 min before fixation, permeabilized, and incubated with Alexa Fluor‐conjugated secondary antibody. For pre‐fixation permeabilization, cells were washed with DPBS and permeabilized with 0.0025% saponin in 10 mM MES, pH 6.1, containing 138 mM KCl, 3 mM MgCl₂, 2 mM EGTA, and 320 mM sucrose for 1 min at 37°C. Cells were gently washed with DPBS and fixed with 4% PFA for 20 min. Immunostaining of endomembrane PI(4,5)P₂ was performed as previously described (Hammond et␣al, 2009 (link)), except that PBS was used as buffer and 1% BSA for blocking. Briefly, cells were fixed, permeabilized with 20 μM digitonin for 5 min, blocked with 1% BSA, and stained with PI(4,5)P₂ antibody. Endomembrane pFAK antibody staining was performed in the same manner as endomembrane PI(4,5)P₂ staining.