Refrax mounting medium

MTT reagent Thiazolyl Blue Tetrazolium Bromide (Sigma Aldrich), All cell culture dishes (Nunc), TRIzol (Ambion), DEPC, Diethyl pyrocarbonate (Ambion), High-Capacity Reverse Transcription Kit (Applied Biosciences, Inc. Foster, CA), DyNAmo ColorFlash SYBR Green qPCR kit (Thermo Scientific), EDTA-free Protease-cocktail inhibitor (Roche Mannheim Germany), Agarose (Invitrogen by Life Technologies), Crystal violet (Sigma-Aldrich), Gelatin (Merck), PFA, Paraformaldehyde (Merck), Mouse monoclonal Anti-N, Nucleocapsid protein of MHV-JHM (monoclonal clone 1-16-1, kindly provided by Julian Leibowitz, Texas A&M, College Station, TX), Anti-CD11b (Abcam; catalog no. ab133357), Anti-Iba1 (Wako, Richmond, VA, USA, Cat no. 019-19741, RRID:AB_839504) antibody, Avidin-biotin immunoperoxidase technique (Vector Laboratories), Refrax mounting medium (Anatech Ltd., MI, USA), Direct-Zol RNA MiniPrep (Zymo Research), Turbo DNA-Free Kit (Life Technology), High Fidelity cDNA Synthesis Kit (Roche), Fast Start Universal Probe Master (Rox) (Roche), Viral-ToxGlo assay (Promega), Prime-direct probe RT-qPCR mix (Takara).

Mice were perfused transcardially with PBS followed by PBS containing 4% paraformaldehyde (PFA) at day 5 p.i. Brain tissue was collected, post-fixed in 4% PFA overnight, and embedded in paraffin [28 (link)]. Brains were sectioned at 5 μm and stained with hematoxylin and eosin (H&E) to evaluate inflammation or meningitis. The paraffin-embedded slides were first deparaffinized on a hot plate followed by xylene treatment for 10 mins. Sections were then rehydrated gradually through different alcohol grades from 100 to 50%. Sections were then treated with hematoxylin for 1 min and excess stain was washed under running tap water. Subsequently, sections were dehydrated by passing through 50 to 95% ethanol. Sections were then stained with eosin stain for 30 s and washed with 95% ethanol twice. Further dehydration was carried out with 100% ethanol and xylene. Sections were mounted with Refrax mounting medium (Anatech Ltd., MI, USA) and observed under the upright light microscope (Nikon Eclipse 50i) and analyzed with Nikon imaging software (NIS, Nikon Corp. Tokyo, Japan).