Histology

Liver specimens were fixed for 24 hours at room temperature in 4% formaldehyde-PBS solution and paraffin embedded. After cutting, sections (3–4 μm) were deparaffinized using xylene and ethanol, stained with hematoxylin and eosin and a trained pathologist analyzed (blinded) liver pathology using a scoring system, considering lobular and portal inflammation as well as endothelitis and thrombus formation. Each parameter was scored from 0 to 4 points, with 0 representing absent, 1 very mild, 2 mild, 3 moderate and 4 severe. For staining of cleaved caspase 3 and LCMV NP, antigen-retrieval was performed using citrate buffer pH6.0 (Vector laboratories) followed by blocking of endogenous peroxidase with 2% H₂O₂ and blocking steps using an avidin biotin blocking kit (Vector laboratories) and rabbit serum. Thereafter sections were incubated over night in either anti-cleaved caspase 3 antibody (Cell signaling) or anti-LCMV nucleoprotein rabbit serum. On the next day, the slides were washed with PBS and incubated with biotinylated swine anti-rabbit IgG (Dako) and developed using the Vectastain ABC kit (Vector laboratories) and DAB-HRP conjugate (Vector laboratories). After counterstaining with hematoxylin and embedding, the sections were analyzed by light microscopy. From each mouse 10 random pictures were taken with a 20-fold magnification and positive cells/field were counted by eye. For the LCMV positive cells, large polyglonal hepatocytes were distinguished from small spindle-shaped infected cells, which were considered as Kupffer cells⁶¹.