Quantifying Zika Virus Impact on Organotypic Hippocampal Cultures

OHC were immune stained as previously described [27 (link)]. The density of mature neurons in OHC 8 h, 24 h, 48 h, 5, 7 and 10 days post infection (p.i.) was evaluated using Cy3-conjugated anti-NeuN antibody (1:100, MAB377C3, Merck or 1:100, ABN78C3, Merck). The presence of ZIKV NS1 protein in the OHC was determined 8, 24 and 48 h p.i. with the unconjugated anti-NS1 antibody (1:1000), kindly provided by Dr. Ada Alves from Oswaldo Cruz Institute, Fiocruz. Activated microglia morphology and density in the OHC were estimated 8 h p.i. with the unconjugated anti-Iba1 antibody (1:100, AB178847, ABCAM, Cambridge, UK). Also, the histone H3 trimethylated in the lysine 4 (H3K4me3) was quantified 5, 7 and 10 days p.i. with the unconjugated anti-H3K4me3 antibody (1:500, 05-1339, Merck). Unconjugated antibodies were subsequently labeled with anti-rabbit (1:400, A11034, ThermoFisher, Waltham, MA) or anti-mouse Alexa Fluor 488-conjugated anti-IgG antibody (1:20,000, A10684, ThermoFisher). All OHC were counterstained with 4′,6′-diamino-2-phenyl-indole (DAPI) (1 µg/mL, D1306, ThermoFisher) and slides were mounted with ProLong Diamond Antifade (P36961, ThermoFisher).