IHC was done as previously described (25). Briefly, mice were perfused and dissected brains fixed with 4% formaldehyde, embedded in paraffin, and sections cut at 7 μm thickness. Before staining, sections were deparaffinized and rehydrolyzed, and antigens were retrieved for 30 minutes at 80°C using the Target Retrieval Solution (Darko). When using cells on coverslips, the cells were fixed for 10 minutes with 4% formaldehyde and permeabilized in 0.2% Triton X-100 for 5 minutes. The samples were blocked with 1% BSA in TBS with 0.3% Triton X-100 (TBS-TX) for 1 hour at room temperature (RT). Samples were stained by incubation overnight at 4°C with the following primary antibodies: mouse monoclonal anti–HSV-1 VP5 (1:300; Abcam, clone 3B6), rabbit polyclonal anti–HSV-1 (1:500; Dako Cytomation, B0116), rabbit polyclonal anti–cleaved caspase-3 (1:300; Cell Signaling Technology, Asp175), goat polyclonal anti-Iba1(1:400; Abcam, ab5076), mouse monoclonal anti-NeuN (1:300; Millipore, clone A60), mouse monoclonal anti-GFAP (1:400; Sigma-Aldrich, clone G-A-5), rabbit monoclonal anti-GSDMD (Abcam, EPR19828), rabbit polyclonal anti–Phospho-RIPK3 (Cell Signaling Technology, Thr231/Ser232), rabbit polyclonal anti-MLKL (Abcam, ab172868), or mouse anti–cytochrome c (1:200; Abcam, ab65311). As control for staining, we used secondary antibody alone or isotype control if the primary antibody was monoclonal. For identification of PIpos cells in the brain tissues, PI (200 μL of 1μg/μL PI solution) was i.p. administered to mice at 90 minutes before perfusion and fixation of the mice. TUNEL staining was done on brain sections using the Apoptag S7111 kit (Millipore) according to the manufacturer’s instructions. After several washes in TBS-TX, the appropriate secondary antibodies coupled to Alexa Fluor 647, 568, or 488 (1:500; Invitrogen) were incubated for 1 hour at RT. Nuclei were stained with DAPI for 6 minutes. Finally, the sections were washed 5 times with TBS-TX and mounted with ProLong Gold. Sections were imaged on a Zeiss LSM 710 or LSM 800 laser scanning microscope or Leica Leitz DMRB fluorescence microscope. Zen 2012 acquisition software and ImageJ (NIH) were used for imaging and analysis.

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