Kpl trueblue peroxidase substrate

A SARS-CoV-2 strain (GISAID# EPI_ISL_425177) was received from the Vaccine and Infectious Disease Organization at the University of Saskatchewan and used as a positive control and plaque reference. Like the clinical isolates described in this report, this strain formed plaques that exhibited a halo-like appearance on Vero CCL-81 cells under a CMC overlay. To further confirm the identity of a subset of virus isolates, the fixed and strained plates were de-stained with ethanol and then immunostained with a 1:500 diluted rabbit anti-SARS-CoV-2 spike antibody (ProSci). The plaques were then visualized with a 2° goat anti-rabbit IgG antibody conjugated to horseradish peroxidase (Invitrogen) and a KPL TrueBlue peroxidase substrate (SeraCare).

Confluent monolayers of Vero-E6 cells in 12-well plate format were infected with tenfold serially diluted samples in 1X phosphate-buffered saline (PBS pH 7.0; Corning 21040CV) supplemented with bovine serum albumin (BSA; MP-Bio 810063) and penicillin–streptomycin for an hour while gently shaking the plates every 15 min. Afterwards, the inoculum was removed, and the cells were incubated with an overlay composed of MEM with 2% FBS and 0.05% purified agar (OXOID-UK; LP0028) for 72 h at 37 °C with 5% CO₂. The plates were subsequently fixed using 4% formaldehyde overnight and the formaldehyde was removed along with the overlay. Fixed monolayers were blocked with 5% milk in Tris-buffered saline with 0.1% tween-20 (TBS-T) for an hour. Afterwards, plates were immunostained using a monoclonal antibody cocktail against SARS-CoV-2 Spike (Creative-Biolabs; 2BCE5) and SARS-CoV-2 nucleocapsid protein NP (Creative-Biolabs; NP1C7C7) at a dilution of 1:1000 followed by 1:5000 anti-mouse IgG monoclonal antibody and was developed using KPL TrueBlue peroxidase substrate for 10 min (Seracare; 5510-0030). After washing the plates with distilled water, the number of plaques were counted. Data shown here is derived from three independent experimental setups. EMCV samples were processed in VERO-CCL81 cells in 6 well format, incubated for 48 h, and developed with crystal-violet.

crystal violet-based plaque assays were performed to quantify infectious WNV_KUN and ZIKV particles, while immuno-focus plaque assays were performed to quantify infectious LGTV. In brief, series of virus dilutions in DMEM were used to infect 90% confluent Vero cells for 1 h at 37 °C, followed by cell-overlaying with DMEM supplemented with 1.2% Avicel (FMC, Philadelphia, PA, USA), 2% HI‒FBS, 1X NEAA, 1% PEST. After 3–4 days, the overlays were removed, and cells were fixed with methanol for 20 min before performing plaque assays. For immuno-focus assay, the fixed cells were blocked by 2% BSA for 10 min before being labeled with 1:1000 mouse LGTV E, then 1:100 VisUCyte anti-mouse secondary HRP Polymer for 1 h at 37 °C, respectively. Finally, the cells were incubated with KPL TrueBlue Peroxidase Substrate (Seracare, Milford, MA, USA) for 20 min at room temperature (RT). For crystal violet-based plaque assay, the fixed cells were stained with 1% crystal violet (Sigma), 20% methanol (Fisher, Trinidad and Tobago), and 1% ammonium oxalate (Sigma) solution.

For determination of viral load in mouse lung tissues a standard plaque assay was performed. Confluent monolayers of MDCK cells were infected with serial dilutions of homogenized lung tissue ranging from 1:10 to 1:1,000,000 diluted in 1X MEM (1% penicillin–streptomycin antibiotics mix, 1% HEPES, 1% l-glutamine and 1% sodium-bicarbonate (Gibco)) for 1 h at 33 °C, with shaking every 15 min. Afterwards, an overlay containing 2% Oxoid agar (Thermo Fisher), H₂O, 2X MEM, DEAE and TPCK-treated trypsin was added to the cells. The plates were incubated at 33 °C for 3 days and then fixed with 3.7% paraformaldehyde overnight at 4 °C. Plaques were visualized by immunostaining. Here, the agar overlay was removed and the plates blocked with 3% milk and PBS. The blocking solution was removed and primary antibody ((H1N1 guinea pig anti-sera (generated in house)) diluted 1:3,000 in 1% milk and PBS was added for 1 h. The plates were washed three times with PBS and secondary antibody (anti-mouse IgG H&L peroxidase-conjugated (Rockland) diluted 1:3,000 in 1% milk and PBS was added for 1 h. The plates were washed three times with PBS and developed by using KPL TrueBlue Peroxidase Substrate (SeraCare).