Metal enhancer tablets

The protocol was performed as previously described, with slight modifications (Hung et al., 2016 (link)). Vibratome sections of fly tissue were fixed in 2% glutaraldehyde in buffers containing 0.1 M sodium cacodylate with 2 mM CaCl₂, pH 7. Residual glutaraldehyde was washed off (five washes for 2 min each) and quenched with 20 mM glycine followed by five more washes. The specimens were subsequently stained with SIGMA FAST™ DAB (3,3′-diaminobenzidine tetrahydrochloride) with Metal Enhancer Tablets (Sigma-Aldrich) for 20 min, washed in the buffer (10 min each, five times), and stained with 1% osmium tetroxide for 30 min. After washing with ddH2O (10 min each, three times), the specimens were stained with 1% uranyl acetate overnight. The specimens were then further dehydrated and embedded in resin for thin-sectioning and TEM observation.
TFAM-Apex2 staining signals were analyzed using Fiji ImageJ software. First, Gaussian Blur filters were applied to the TEM images, and then, the threshold was adjusted to isolate TFAM-Apex2 signals. The intensities and area of individual TFAM-nucleoids were plotted in box-and-whisker graphs. Statistical analyses were performed by the F-test and t-test functions in Excel.

To monitor the purification process and confirm rgD5 antigenicity prior to the ELISA development, SDS-PAGE was carried out on 12% polyacrylamide gel. The gels were either stained overnight with Coomassie Blue R-250 (Bio-Rad) or electroblotted onto nitrocellulose membrane (Bio-Rad) using Bio-Rad Mini Trans-Blot Cell (Bio-Rad). Briefly, the membrane was blocked with 5% non-fat milk and, after three washes in phosphate buffer saline containing 0.05% Tween-20 (PBS-T) (137 mM NaCl, 2.7 mM KCl, 100 mM Na₂HPO₄, 2 mM KH₂PO₄, pH 7.4), the membrane was incubated with mouse monoclonal antibody (MAb) anti-6xHis HRP conjugated (Invitrogen) or with polyclonal antibody (PAb) anti-BoHV-5 from bovine experimentally immunized with inactivated BoHV-5 as previously described [22 (link)]. The immunoblot was developed using Sigma FAST 3,3’-Diaminobenzidine (DAB) with Metal Enhancer tablets (Sigma). The protein concentration was determined by bicinchoninic acid (BCA) protein assay (Pierce) method according with the manufacturer’s instructions.