Prolong gold antifade

NRK-49F fibroblasts were seeded on coverslips to a confluence of 50–60% in 24-well plates and infected with wild-type, ∆mrdA and ∆PBP2_SAL strains. Infected cells were fixed at 2, 4, 8 and 24 hpi in 3% PFA (15 min, RT), and processed for immunofluorescence microscopy, as described^{70 (link)}. Briefly, after PFA fixation, the infected cells were washed in PBS pH 7.4 and incubated for 10 min at RT in blocking solution containing 0.1% (w/v) saponin and 1% (v/v) goat serum. Incubations with primary and secondary antibodies were performed in this same solution of 0.1% (w/v) saponin and 1% (v/v) goat serum during 45 min each, with three washes in PBS pH 7.4 after the respective incubations. Coverslips were finally mounted on slides using ProLong Gold Antifade (Molecular Probes). Rabbit polyclonal anti-S. Typhimurium LPS (cat. no. 229481, 1:1000, Difco Antiserum-BD Diagnostics, Sparks, MD) and goat polyclonal anti-rabbit IgG conjugated to Alexa 488 (1:1000, cat. no. A-11008, ThermoFisher Scientific), were used as primary secondary antibodies, respectively. Images were acquired on an inverted Leica DMI 6000B fluorescence microscope with an automated CTR/7000 HS controller (Leica Microsystems) and an Orca-R2 charge-coupled-device (CCD) camera (Hamamatsu Photonics).

3 to 5×10⁴ HeLa cells were plated in eight-well chambered glass slides and infected the next day with wild-type PsV at MOI 20 or mutant PsV containing the same number of reporter plasmid genomes. (The lower sensitivity of immunofluorescence or PLA [see below] compared to reporter gene expression necessitated an MOI of 20 or 50 to visualize virus components.) Eight hours post-infection, the cells were fixed for 15 min at room temperature with 4% paraformaldehyde (Electron Microscopy Sciences, #15710), washed with PBS and then permeabilized with 0.5% Triton X-100 for 20 min at room temperature in PBS. The cells were blocked for one hour in 1% bovine serum albumin and 3% goat serum, and immunostained with 1:200 33L1–7 (obtained from Martin Sapp (LSU)). After extensive washes, AlexaFluor 594-conjugated goat anti-mouse secondary antibodies were added at 1:300 dilution for 40 minutes at room temperature. Nuclei were stained with 1:100 dilution of 4′,6-diamidino-2-phenylindole (DAPI), cells were washed extensively, and slides were mounted in Prolong gold anti-fade (Molecular Probes). Images were recorded on a ZEISS Axiovert 200 inverted fluorescent microscope using appropriate filters processed with ImageJ.

The hemolymph was collected from control and WSSV-injected shrimps at 6, 24 and 48 hpi, as well as from moribund shrimps, and immediately fixed by incubation in 4% (w/v) paraformaldehyde at room temperature for 10 min. The fixed hemocytes were washed in PBS (centrifugation stage at 800×g at 4°C for 10 min) and resuspended in PBS. About 10⁶ hemocytes were attached onto each SuperFrost microscope slide by centrifugation at 1000×g for 10 min. Slides were blocked in 10% (v/v) fetal bovine serum in PBS at room temperature for 1 h and then probed with purified rabbit polyclonal antibody specific to PmVRP15 and purified mouse monoclonal antibody specific to VP28 (WSSV capsid protein) for 1 h at room temperature and washed three times in 0.05% (v/v) Tween-20 in PBS to remove non-specific binding. The Alexa 488-conjugated goat anti-rabbit IgG and Alexa 568-conjugated goat anti-mouse IgG secondary antibodies (Invitrogen) were applied to the slides and incubated at room temperature for 1 h. The slides were then washed three times as above, incubated with TO-PRO-3 iodide (Molecular Probes) to stain the nuclear DNA and then washed once with PBS. Mounting medium, ProLong Gold antifade (Molecular Probes), was applied and the slides were examined by CLSM (Olympus). Bright field and fluorescence images were collected for the analyses.

Rat kidneys were harvested at approximately 8 weeks post-infection and immediately fixed by immersion in neutral buffered 10% formalin, processed routinely, embedded in paraffin, cut into 4 μm sections, and stained with hematoxylin and eosin (HE). Immunohistochemistry was performed on paraffin-embedded tissue sections using antiserum generated against outer membrane vesicles (OMV) of Leptospira species or with anti-LipL32 (Nally et al., 2004 (link), 2017 (link)). After dewaxing, tissue sections were blocked with 10% normal goat serum in PBS for 30 mins at room temperature. Samples were incubated with anti-OMV or anti-LipL32 at 1:200 in blocking solution and incubated overnight at 4°C. After 3 × 5 min washes in PBS, samples were incubated in goat anti-rabbit IgG conjugated to AlexaFluor 546 (Invitrogen, CA) and DAPI (Invitrogen, CA) 1:3,000 in blocking solution for 60 min at room temperature in the dark. Samples were again washed in PBS before the addition of ProLong Gold anti-fade (Molecular Probes, OR) mounting media per slide and covered with a 24 × 50 mm coverslip. Samples were viewed using a Nikon Eclipse E800 and images captured using Nikon Elements Software.