Vector vip hrp substrate kit

The ZIKV^PR strain, PRVABC59, was obtained from BEI resources and propagated in C6/36 mosquito cells in serum-free neuro-glial differentiation media (62 (link)). This protocol was adapted from standard techniques (63 (link)) for infections in neural models and produces high titer virus. Virus-containing cell supernatants were harvested after the appearance of cytopathic effect in the C6/36 cells and clarified by centrifugation. Zika-conditioned media was collected from uninfected C6-36 cells grown in parallel. Viral titer was determined by focus-forming assay to calculate BHK-21 focus-forming units: BHK-21 cells (ATCC) were infected with dilutions of viral stock and overlaid with 3.2% carboxymethylcellulose solution mixed 1:1 with Dulbeccos modified Eagle medium (DMEM) containing 2% Fetal Bovine Serum (FBS) (63 (link)). At 4 dpi, cells were fixed and stained with 5 ug/mL anti-ZIKV NS1 protein monoclonal antibody #110 (64 (link)), followed by secondary staining with Horseradish peroxidase (HRP)-conjugated goat-anti-mouse antibody (Promega) and detection using the Vector VIP HRP substrate kit (Vector Laboratories).

Cell culture media was obtained from Lonza. FBS and trypsin-EDTA were obtained from Life Technologies (Paisley, UK). Chick embryo extract was purchased from Sera Labs International (Sussex, UK). Phospho-AMPK^Thr172 (40H9) and total AMPKα (F6) antibodies were obtained from New England Biolabs (Herts, UK). Anti-myosin, skeletal fast (clone MY-32) and β-actin (clone AC-15) antibodies were purchased from Sigma. Monoclonal mouse anti-human desmin (D33) antibody was obtained from DAKO. Vector VIP HRP-substrate kit was obtained from Vector Laboratories. 2-Deoxy-D-[2,6-³H]glucose was purchased from Hartmann Analytic (Germany). IL6 ELISA kits were obtained from Qiagen (Sussex, UK).

Muscle cell origin was confirmed immunohistochemically using antibodies to the muscle-specific protein desmin. Cells were fixed and permeabilised by incubation in ethanol at 4°C overnight before washing with phosphate-buffered saline (PBS) and blocking in PBS containing 2% (v/v) FBS. Cells were incubated in anti-desmin antibody at a 1:100 dilution in PBS/FBS 2% for 1hr at room temperature. After washing with PBS and a further incubation in PBS/FBS 2%, rabbit anti-mouse HRP-conjugated secondary antibody was added at a 1:300 dilution for 1hr at room temperature. After washing with PBS, Vector VIP HRP-substrate kit was used to develop the reaction product.