FFPE brain specimens of 5 deceased patients with HSE and 1 control patient who died of severe head trauma were obtained postmortem for diagnostic purposes. According to the institutional “opt-out” system (Erasmus Medical Centre), which is defined by the National Code of Good Conduct (Dutch: Code Goed Gebruik, May 2011), these surplus human brain tissues were available for the current study. Sections of 4 μm thickness were cut from the human FFPE brain biopsies and cooked at 60°C for 1 hour prior to being deparaffinized. Antigen retrieval was performed with citric acid for 15 minutes and TrueBlack Lipofuscin Autofluorescence Quencher (Biotium) was applied to limit background fluorescence. TUNEL staining was done on brain sections using the Apoptag S7111 kit (Millipore) according to the manufacturer’s instructions. Subsequently, sections were treated with 3% H2O2 and 1% absolute methanol for 30 minutes and blocked with 0.1% BSA diluted in TBS before staining with the following primary antibodies: polyclonal rabbit anti-GFAP (1:10000; Dako, Z0334), polyclonal rabbit anti-Iba1 (1:500; Wako, 019-19741), rabbit monoclonal MLKL (phospho S358; 1:250; Abcam, EPR9514), mouse monoclonal anti-GSDMD (1:1000; Abnova, 3F12-1B2), polyclonal rabbit anti–cleaved caspase-3 (1:50; Promega, G7481), monoclonal mouse anti–HSV-1 (1:50; Cell Marque, 10A3), or monoclonal mouse anti-CD45 (1:50; Dako, 2B11 + PD7/26). After overnight incubation with the primary antibodies in TBS-TX with 0.1% BSA, sections were washed extensively with TBS-TX and subsequently incubated for 1 hour at RT with the appropriate secondary antibodies: Alexa Fluor 647 goat anti-rabbit (1:250; Invitrogen, A21244) or Alexa Fluor 555 goat anti-mouse (1:250; Invitrogen, A21127). Finally, sections were incubated with Hoechst dye (1:1000; 20 mM 33342 solution, Invitrogen) for 10 minutes at RT to visualize nuclei and mounted with ProLong Diamond Antifade Mountant for imaging using a Zeiss LSM 700 confocal microscope as described previously (63).

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