Lsm 710

All mice were euthanized at 68 to 70 weeks of age for histological and immunological analyses. As previously reported [18 (link)], we stained and examined whole mounts of adipose tissue. Mice were sacrificed by cervical dislocation, after which the VAT was removed by a sterile technique and minced into small pieces (~2–3 mm) with a scalpel. The tissue pieces were washed, fixed in cellFIX (Cat. 340181, BD) for 60 min, and permeabilized with 0.1% Triton X-100 for 10 min. Then the specimens were blocked with 5% bovine serum albumin, incubated with the primary antibody [F4/80 (BM-8, eBioscience, San Diego, CA, USA)] overnight at 4°C, and then incubated with the Alexa Fluor 488-conjugated secondary antibody (Molecular Probes, Eugene, OR, USA) for 1 h. The tissues were counterstained for 1 h with BODIPY 558/568 (Molecular Probes) to visualize adipocytes and with 4',6-diamidino-2-phenylindole (DAPI; Molecular Probes) to visualize nuclei. For confocal microscopy (LSM 710, Carl Zeiss, Jena, Germany), the tissue samples were illuminated with four laser lines (405 nm, 488 nm, 568 nm, and 800 nm) and emissions were collected through appropriate narrow band-pass filters, after which images were acquired and processed by LSM 710 software.

For microscopy, J774A.1 cells were incubated with strain H2 (MOI, 0.5) for the different times in 35-mm culture dishes containing Opti-MEM medium. To examine the integrity of cellular structure, PI (2 μg/mL) was added to the culture. After incubation for 5 min, the cells were observed with a confocal microscope (Zeiss LSM 710, Germany). To examine lysosomal membrane permeability, cells were stained with Acridine Orange (2 μg/mL) at 37°C for 15 min and then washed with PBS. Images were captured using a confocal microscope (Zeiss LSM 710, Germany).

To visualize leptin expression, 5 μm-thick frozen RPV sections were fixed using 4% formaldehyde and permeabilized using 0.2% Triton X-100. Nonspecific binding was blocked by 1% BSA, 0.1% Triton x-100 in PBS for 10 min followed by incubation with anti-leptin antibody [Ob (Y-20) at 1:100 ratio in 1% BSA and 0.05% Tween in PBS] for 1 h. RPV sections were then washed and incubated for another hour at room temperature with Alexa 594-conjugated goat anti-mouse secondary antibody (1:250 in 1% BSA and 0.05% Tween in PBS; Molecular Probes). Images were acquired with a laser confocal microscope (LSM710, ZEN confocal software Carl Zeiss).
For F-actin and G-actin ratio study, RPV frozen sections were fixed in 4% formaldehyde, 0.2% Triton x-100 in the cytoskeleton stabilizing PEM buffer (100 mM PIPES, 5 mM EGTA, 2 mM MgCl2, pH 6.9) for 20 min at room temperature. Thereafter, the sections were permeabilized, blocked, and stained with the F-actin stain Phalloidin (100 nM; Acti-stain 555 phalloidin, Cytoskeleton, Denver, CO, USA) and the G-actin stain Deoxyribonuclease I (300 nM; Alexa Fluor 488 conjugate, Invitrogen, NY, USA). All sections were examined and positive intensity signals were quantified using a laser confocal microscope (LSM710, ZEN confocal software Carl Zeiss).

Vero cells grown on microscope coverslips in 6-well tissue culture plates were mock-infected or infected with PEDV. Assessment of salinomycin effects on PEDV infection of cells involved pretreating the cells with salinomycin at concentrations of 0.01, 0.1 and 1 μm before infecting them with PEDV. Consequently, cells were fixed in 4% paraformaldehyde for 10 min at room temperature (RT) and permeabilized using 0.2% Triton X−100 in PBS at RT for 10 min. Then, they were blocked using 0.4% bovine serum albumin (BSA) in PBS for 30 min at RT after which they were incubated with PEDV N-specific MAb for 2 h. They were washed 5 times using PBS, re-incubated with a goat anti-mouse secondary antibody conjugated to Alexa Fluor 488 (Invitrogen) for 1 h at RT, then counterstained with DAPI (Sigma). Coverslips were mounted on the microscope glass slides in a mounting buffer. Cell stains were observed using a confocal laser microscope (LSM-710; Zeiss, Oberkochen, Germany) and visualized by CLSM (LSM 710, Zeiss, Oberkochen, Germany).