R9759

Immunofluorescence was used to identify cells expressing ISG15 and PKR as described [49 (link), 50 (link)]. Briefly, after citrate buffer heat-mediated antigen retrieval and incubation with PBS containing 20 % horse serum or 20 % goat serum to block non-specific binding, paraffin sections were co-incubated with antibodies directed against ISG15 (sc-50366; 1:100) or PKR (ab-32036; 1:200) and antibodies directed against GFAP (ab-3554, Abcam; 1:200), Olig2 (ab-85900, Abcam; 1:500), or CD107b (MCA2293, AbD Serotec, Puchheim, Germany; 1:200) overnight at 4 °C. Negative controls included sections incubated with rabbit serum (R4505, Sigma-Aldrich; 1:3000/1:6000), goat serum (I9140, Sigma-Aldrich; 1:4500), sheep serum (1:5000), and rat serum (R9759, Sigma-Aldrich; 1:16000), respectively. After washing, sections were incubated Cy2- and Cy3-conjugated secondary antibodies (goat anti-rabbit, A-11034, Invitrogen; 1:200; goat anti-rat, 112-165-003; donkey anti-rabbit, 711-545-152; donkey anti-sheep, 713-765-147; donkey anti-goat, 705-765-147, Jackson ImmunoResearch, Suffolk, UK; 1:200) in the dark for 1 h at room temperature. Nuclei were counterstained with 0.01 % bisbenzimide (H33258, Sigma-Aldrich), and sections were mounted in Dako Fluorescence Mounting Medium (S3023, DakoCytomation GmbH, Hamburg, Germany).