Ab3554

Mice were anesthetized with sevoflurane, and brains were cut on a sliding freezing stage microtome in 30 μm thick sections. Hippocampal sections were washed 3 times in PBS. Following 15 min permeabilization with 0.3% Triton X-100 in 0.01 M PBS, sections were blocked with 10% donkey serum for 2 h. Next, sections were incubated overnight at 4°C with the following primary antibodies: DHX9 (1:300, 17721-1-AP, Proteintech), NeuN (1:200, ab104224, Abcam), Iba1 (1:200, ab5076, Abcam), GFAP (1:200, ab3554, Abcam), MAP2 (1:200, ab11267, Abcam), FMRP (1:100, sc-101048, Santa Cruz), STAU1 (1:100, sc-390820, Santa Cruz), or c-Fos (1:200, ab208942, Abcam). The next day, slides were washed 3 times for 7 min in PBS. Then, sections were incubated with the corresponding fluorescent-conjugated secondary antibody (A32766, A32744, A32790, A32754, Invitrogen) for 2 h at room temperature in the dark. DAPI Fluoromount-G aqueous mounting medium with anti-fade properties (SouthernBiotech) was used to identify cell nuclei. Slides were stored in the dark at 4°C until needed for analysis. Fluorescence images were captured with an Olympus FV1000 laser confocal microscope. For c-Fos positive neuron analysis, ImageJ was used to analysis the signals of hippocampal neurons from 3 animals per condition.

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).