In vivo imaging system fx

The methods used have been described previously (Zheng et al., 2014 (link)). Following a 24-h fast, a 2.5 ml barium meal was administrated to each rat through oral gavage. Plain radiographs of the GI tract were obtained using a KODAK in vivo Imaging System FX with the focus distance manually fixed to 50 ± 1 cm. The exposure time was adjusted to 30 s. Images were recorded at different time points (30, 60, and 90 min) after the consumption of the barium meal. Gastric emptying (GE) was calculated according to the following formula:

Vero E6 cells were seeded overnight into 12-well plates to achieve an 80% confluent monolayer the next day. Viral culture supernatants collected for LCMV titration were centrifuged to remove cell debris, tenfold serially diluted in DMEM supplemented with 2% FCS and added to Vero E6 cells. After 90 min viral adsorption period, the inoculum was aspirated, cells were washed with PBS and overlaid with carboxymethyl cellulose in DMEM supplemented with 5% FCS. Three days after infection, the cells were fixed in ice-cold methanol for 20 min at − 20 °C, washed with PBS, and blocked with 3% BSA in PBS for 1 h. After blocking, the cells were incubated for 1 h with an antibody against LCMV NP (M87). Subsequently, the cells were washed 3× with PBS and incubated for 45 min with peroxidase-conjugated goat anti-mouse antibody (dilution 1:10,000 in blocking buffer), then washed 3× with PBS and subjected to chemiluminescent detection using In-Vivo Imaging System FX (Kodak). LCMV titer was expressed as focus forming units per milliliter (FFU/ml).

Hind limbs were stripped of connective tissues and fixed in ethanol (70% v/v) prior to radiological assessments. Radiographs of the mouse paws were obtained using a Kodak In vivo Imaging System FX, and images were assessed using Kodak Molecular Imaging software (Kodak Molecular Imaging Systems, Connecticut, USA) as described previously [29 (link)]. A radiographic score was established for each limb by counting the number of eroded joints. The central/intermedium, fourth to fifth distal tarsals/fibulare, first to fifth metatarsal/tarsal and the first to fifth phalangeal/metatarsal joints were counted (score = 1 for each eroded joint and maximum score = 12 for each limb). Two independent musculoskeletal clinicians who were blinded to the CIA protocols scored the radiographs.