Goat anti mouse igg2a alexa fluor 546

For all staining experiments, WT MOVAS cells and KO MOVAS cells were seeded into 4-chamber tissue culture slides (Corning) and grown to optimal confluency before being washed with PBS and beginning individual respective treatments which involved the following: (1) incubated in SFM with vehicle for 72 hours; (2) incubated in SFM with 10 μg/mL of MβCD:Chol for 72 hours; (3) incubated in SFM with 10 μg/mL of MβCD:Chol for 72 hours, washed with PBS, and exposed to efflux medium for 72 hours. For cellular stain preparations, MOVAS cells were prepared, stained, and imaged as described [38 (link)], with this work being conducted at the Clemson Light Imaging Facility (CLIF). Briefly, cells were incubated with ACTA2 (sc-32251; Santa Cruz Biotechnology) and CD68 (sc-20060; Santa Cruz Biotechnology) primary antibodies, then Alexa Fluor 546 goat anti-mouse IgG_2a (Invitrogen, Carlsbad, CA, USA) and Alexa Fluor 488 goat anti-mouse IgG₁ (Invitrogen) secondary antibodies. Nuclear counterstaining was performed using DAPI (Invitrogen). Imaging was performed with a Leica SP8X MP Confocal System equipped with HyD detectors, a 405 nm laser, a tunable white light laser, and time gating capabilities (Leica Microsystems, Buffalo Grove, IL, USA). For imaging capture/export, a Leica LAS-X software (Leica Microsystems Version 3.5.5.19976) was used [38 (link)].

DAPI (dilution 1:1000, Thermo Fisher Scientific, 62248), Goat anti-Hamster IgG Alexa Fluor 647 (dilution 1:200, Jackson ImmunoResearch, 127-605-160), Goat anti-Rabbit IgG Alexa Fluor 430 (dilution 1:200, Thermo Fisher Scientific, A11064), Goat anti-Rabbit IgG Alexa Fluor 430 (dilution 1:200, Thermo Fisher Scientific, A11064), Goat anti-Rabbit IgG Alexa Fluor 594 (dilution 1:200, Thermo Fisher Scientific, A11037), Goat anti-Mouse IgG1 Alexa Fluor 488 (dilution 1:200, Thermo Fisher Scientific, A21121), Goat anti-Mouse IgG1 Alexa Fluor 647 (dilution 1:200, Thermo Fisher Scientific, A21240), Goat anti-Mouse IgG2a Alexa Fluor 546 (dilution 1:200, Thermo Fisher Scientific, A21133), Goat anti-Mouse IgG2b Alexa Fluor 647 (dilution 1:200, Thermo Fisher Scientific, A21242), Goat anti-Guinea Pig IgG Alexa Fluor 546 (dilution 1:200, Thermo Fisher Scientific, A11074), Goat anti-Guinea Pig IgG Alexa Fluor 594 (dilution 1:200, Thermo Fisher Scientific, A11076), Donkey anti-Chicken IgG IRDye 680LT (dilution 1:200, Li-Cor Biosciences, 926-68028), Donkey anti-Rabbit IgG IRDye 800CW (dilution 1:200, Li-Cor Biosciences, 926-32213), Donkey anti-Chicken IgG Alexa Fluor 488 (dilution 1:500, Jackson ImmuoResearch, 703-545-155), and Donkey anti-Guinea Pig IgG Alexa Fluor 647 (dilution 1:500, Jackson ImmuoResearch, 706-605-148).

DF-1 cells were co-transfected as described above or infected with strain Ct at a multiplicity of infection (MOI) of 0.01. After 24 h, the cells were detached with trypsin–EDTA and washed with PBS 5% FBS, followed by fixation with Fixation buffer, washed twice with Permeabilization buffer (reference 88-8824, ThermoFisher) and incubation with mouse anti-VP3 monoclonal antibody (clone IBDV9, reference 3BD5, Hytest). After three washes with Permeabilization buffer, cells were incubated with a goat anti-mouse IgG2a Alexa Fluor 546 (reference A21133, ThermoFisher), washed again three times with Permeabilization buffer and analyzed with a FC500 MPL flow cytometer (Beckman Coulter). The fixation step and antibody incubations were carried out during 30 min at room temperature in the dark.

DF-1 cells seeded onto glass coverslip in 24-well plates were infected with Ct viral stock at a MOI of 0.01 or co-transfected as described above with 0.4 µg of each plasmid construct expressing segments A or B. After 24 h, coverslips were washed with PBS, fixed with ethanol:acetone solution (1:1 ratio) for 20 min at − 20 °C and dried for 15 min under chemical hood. Coverslips were blocked for 30 min using PBS containing 5% FBS. Later, samples were incubated for 1 h with mouse anti-VP3 monoclonal antibody (clone IBDV9, reference 3BD5, Hytest) diluted in PBS 5% FBS. Coverslips were washed three times with PBS and incubated for 30 min with a goat anti-mouse IgG2a Alexa Fluor 546 (reference A21133, ThermoFisher) and Hoechst 33342 ( reference 14533, Sigma-Aldrich) diluted in PBS 5% FBS. All incubations were performed at room temperature. Finally, coverslips were dried and mounted with ProLong diamond antifade mountant (reference P36965, Thermofisher). Samples were visualized with an Olympus BX41 inverted fluorescence microscope.