A10684

The OHCs were immune-stained according to Gogolla and collaborators [22 (link)] using 0.05% Tween in PBS as permeabilization solution. Mature neurons were labeled with rabbit or mouse antibodies conjugated to the Cy3 fluorophore (Merck, #ABN78C3 or #MAB377C3, 1:100). Activated microglia and trimethylation of different lysine residues of histone H3 were indirectly labeled with primary antibodies anti-Iba1 (Abcam, #AB178847, 1:100), anti-H3K4me3 (Merck, #05-1339, 1:500), anti-H3K9me3 (Merck, #07-442, 1:500) and anti-H3K36me3 (Abcam, #AB9050, 1:200), respectively. As secondary antibodies, anti-rabbit (Thermo Fisher, #A11034, 1:400) or mouse (Thermo Fisher #A10684, 1:20,000) IgG conjugated with Alexa Fluor 488 fluorophore were used.
DNA fragmentation in apoptotic cells was assessed with the TUNEL assay (Thermo Fisher), performed according to the manufacturer’s instructions.
For BrdU detection, cultures were incubated in HCl 1 M for 30 min after permeabilization and stained with mouse anti-BrdU antibody conjugated to Alexa Fluor 488 (Merck, #FCMAB101A4, 1:100).
The OHCs were counterstained with DAPI (Thermo Fisher) and mounted onto glass microscope slides with ProLong Diamond Antifade Mountant (Thermo Fisher).

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).