51 fluorescence microscope

Blood samples were collected and serum was isolated for the measurement of VNA by using the FAVN tests as described previously [41 (link)]. In brief, 50 μl of serial 3-fold dilutions of test and standard serum samples were added to 96-well microplates in 100-μl volumes. Each sample was added to four duplicated chambers. A 50-μl volume of CVS-11 suspension containing 50-200 FFU was added to each chamber. The microplates were then incubated at 37°C for 1 h with 5% CO₂. Then, 50 μl of the BSR cells (5×10⁵ cells/ml) were added into each chamber, and the microplates were incubated at 34°C with 5% CO₂ for 3 days. The plates were fixed in 80% ice cold acetone at -20°C for 30 min and then air-dried. Cells were stained with FITC-conjugated anti-RABV N antibodies for 45 min at 37°C, and then washed three times with PBS. The results were observed using an Olympus 1×51 fluorescence microscope. VNA titers were expressed in IU/ml based on comparisons with the titer of a reference serum obtained from the National Institute for Biological Standards and Control (Herts, UK) in each test.

BSR cells seeded in 24-well plates were non-infected or infected with different rRABVs at MOI of 0.01. Two days later, the cells were fixed for 1 h with 2% paraformaldehyde and permeated for 1 h with 1% Triton X-100. Then, the cells were blocked by 2% fetal bovine serum (FBS). The cells were incubated with rabbit anti-mouse HMGB1 antibody, and subsequently stained with Alexa Fluor 488-conjugated goat anti-rabbit antibody. 4', 6-diamidino-2-phenylindole (DAPI) (1 μg/ml) was used to dye the nucleus. The cells were washed three times with PBS after incubation with each above mentioned antibody. Fluorescent images were captured under an Olympus 1×51 fluorescence microscope.