Evaluating Anti-Inflammatory Compounds in Sharpin-Deficient Mice

The Sharpin^cpdm/cpdm mice were described earlier [38 (link)]. At 4-week of age, Sharpin^cpdm/cpdm and Sharpin^+/+ littermates were fed a standard diet enriched, or not, with 0.7% w/w BHA or BHT (Ssniff, Soest, Germany). After 5 weeks on this diet, the mice were killed and the degree of inflammatory symptoms was assessed. The organs were fixed in 4 % paraformaldehyde, embedded in paraffin, and cut at 3 or 5 µm thickness. Subsequently, sections were stained with hematoxylin and eosin. The terminal deoxynucleotidyl transferase dUTP nick end labeling assay was performed according to the manufacturer’s instructions (In situ cell death detection kit, TMR red—Roche). Micrographs were acquired using a Zeiss Axioscan Z.1 slide scanner (Carl Zeiss, Jenna, Germany) at ×20, ×100, ×200, and ×400 magnification, with a Hamamatsu ORCA Flash4 camera (Hamamatsu Photonics) or AxioCam MRm Rev. 3 FireWire camera, via either Zen 3.1 software or AxioVision 4.5 software from Zeiss. Quantification analysis was performed using a script provided by the VIB Bioimaging Core (Ghent, Belgium) ran on QuPath-0.2.3 software. Serum LDH levels were obtained at UZ-Gent (Belgium) using Cobas 8000 modular analyzer series (Roche Diagnostics, Basel, Switzerland). Interleukin (IL)-6 levels were measured using a Bio-Plex Multiplex immunoassay (Bio-Rad #171304070) according to the manufacturer’s instructions.