Dfc3000 g

For eight-week-old GFAP-Cre; Rosa Green and Periostin-Cre; Rosa Green mouse nerves and bones, images were acquired at 100x magnification on a Nikon Eclipse TE300 fluorescence inverted microscope equipped with an optical camera (Leica DFC 3000G) and analyzed using Leica Application Suite Advanced Fluorescence 3.20.9652. Seven day post-injection images were acquired at 200x magnification on a Nikon Eclipse TE300 fluorescence inverted microscope equipped with an optical camera (Leica DFC 3000G). Images from tumor bearing mice were acquired at 400x magnification using an Olympus BX51 camera.

TMRE staining was performed according to the previous study (Cho et al., 2017 (link)). TMRE was dissolved in DMSO at a concentration of 50 µM and added into fresh bacteria culture at a final concentration of 0.1 µM before seeding the plates. Worms were synchronized by egg bleach and grown on E. coli HT115 for RNAi from the hatch and transferred to RNAi plates containing TMRE at the L3/L4 stage. Worms were imaged after growing overnight on TMRE plates. TMRE staining was quantified with ImageJ. CCCP was dissolved in DMSO at a concentration of 10 mM and added into bacteria culture at a final concentration of 50 µM before seeding the plates. Images were acquired with Leica Application Suite (LAS) using Fluorescent Stereo Microscope Leica M205, with objective lens, PlanApo 5.0× LWD (10447243; Leica), and camera, Leica DFC3000 G, at room temperature.

Three hundred microliters (100μL per well) of haemolymph were deposited on glass slide and centrifuged at 100×g during 1min at 4°C. The supernatant was removed and cells were fixed with ice cold methanol for 30s. After two washes in 1X PBS (5min and 15min, respectively), potential sites of non-specific interaction were blocked using 5% BSA dissolved in 1X PBS during 1h at room temperature. Samples were washed three times in 1X PBS for 10min each. The slides were incubated overnight at 4°C with the two primary antibodies targeting the putative OsHV-1 IAP (1:100) and the actin (1:500) (A4700, Sigma) diluted in 1X PBS supplemented with 1% of BSA. Unbound primary antibodies were removed by three washes in 1X PBS. Primary antibodies were detected using two fluorochrome-conjugated secondary antibodies (1:400) diluted in 1X PBS with 5% of BSA for 45min at room temperature in the dark. The secondary antibodies conjugated with the dylight anti-rabbit 488 (072-03-15-06, Eurobio) and the dylight anti-mouse 549 (DI-2549, Eurobio), were used for viral proteins and oyster actin detection, respectively. After two washes in 1X PBS (5min), samples were mounted using ProLong^® Gold antifade mountant with 4’,6-diamidino-2-phenylindole (DAPI) (P36935, ThermoFisher Scientific). Samples were examined under fluorescence microscope (Leica DFC3000 G).