Kihc 5

The levels of plasma liver enzymes were measured with Roche kits (ALT, AST kits) and Roche biochemical analyzers. Fresh liver tissue samples were fixed in 4% paraformaldehyde in PBS, embedded in paraffin and sectioned at a thickness of 4 μm for hematoxylin & eosin (H&E) staining, TUNEL staining (Cat. No. 12156792910, Roche, Germany) and immunochemistry (KIHC-5, Proteintech, Wuhan, China) according to standard procedures and kit manuals. Histological lesions were scored by a pathologist. We evaluated tissue damage using a semiquantitative method according to the literature^{27 (link)}. For semiquantitative analyses, the necrotic area or lesions in the target region were scored as +1 for <25%, +2 for 25–50%, +3 for 50–75%, and +4 for >75%. In the immunochemistry assay, the antibodies, their dilutions and applications were as follows: anti-LC3A/B, 1:100 (Immunofluorescence, #12741, CST, Boston, USA), goat anti-rabbit IgG H&L (Alexa Fluor^® 488), 1:1000 (Immunofluorescence, ab150077, Abcam, Cambridge, UK), anti-Stx 17, 1:500 (Immunoprecipitation, 17815-1-AP, Proteintech, Wuhan, China), and anti-Vamp 8, 1:10000 (Western blot, ab76021, Abcam, Cambridge, UK).

PBS-perfused brains were fixed in 4% paraformaldehyde, and coronal sections were prepared for IFA (thickness: E18.5, 20 μm; postnatal, 30 μm) or IHC and histochemistry (thickness: 3 μm). Mouse monoclonal antibodies against MCMV IE1 (51 (link)); rabbit monoclonal antibodies against SOX2 (Abcam, ab97959), Tbr1 (Abcam, ab31940, ab183032), Ctip2 (Abcam, ab240636), GFAP (Proteintech, 16825-1-AP), Iba1 (Abcam, ab178847), GSDMD (Affinity, AF4012), or CC3 (Cell Signaling Technology, 9661); and rat monoclonal antibodies against BrdU (Abcam, ab6326) or Ctip2 (Abcam, ab18465) were used in IFA or IHC as indicated. Secondary antibodies for IFA included Alexa Fluor 488 goat anti–mouse IgG1 (Invitrogen, A-21121), Alexa Fluor 568 donkey anti–rabbit IgG (H+L) (Invitrogen, A-10042), Alexa Fluor 647 goat anti–mouse IgG1 (Invitrogen, A-21240), and Alexa Fluor 647 goat anti–rat IgG (H+L) (Invitrogen, A-21247). DAPI (Life Technologies) was used for nuclei counterstaining. Horseradish peroxidase–conjugated antibody (Proteintech, KIHC-5) was used for IHC. IFA, IHC, and histochemistry were performed as described previously (52 (link)).