In: Respiratory22 Jul 2014
After aspirating the culture media, the cells were fixed in the wells for 10 min with 4% paraformaldehyde in phosphate-buffered saline solution (PBS), rinsed with PBS, and harvested by gently scraping with a sterile rubber policeman. Cytospin slides were then prepared from the collected cell suspensions. RSV proteins were detected by indirect immunoperoxidase staining using a primary rabbit polyclonal anti-RSV antibody and a secondary biotinylated goat antirabbit antibody (Dako A/S; Glostrup, Denmark) optimally diluted in TBS (0.05 M Tris-buffered saline solution, pH 7.6) containing 1% bovine serum albumin and 0.1% polysorbate 20 (Tween 20). Unless otherwise indicated, all incubations and washes occurred at room temperature. After blocking endogenous peroxidase with 0.3% H202 in methanol for 10 min, followed by a 30-min incubation with 1% normal goat serum in TBS to prevent nonspecific binding of secondary antibody, the cells were incubated at 37°C for 1 h with anti-RSV antibody (1:100). Following three washes of 5 min each in TBS, the cells were incubated for 60 min with the secondary antibody (1:400), washed as before, and further incubated for 60 min with avidin-biotinylated peroxidase complex (ABC Vec-tastain; Mississauga, ON, Canada). After three washes in TBS, the cells were incubated with metal-enhanced DAB substrate (Pierce; Rockford, IL), counterstained with Mayers hemalun, and mounted in Permount, a toluene-based histological mounting medium (Fisher Scientific; Fairlawn, NJ). Cytospin preparations of RSV-infected or uninfected HEp-2 cells were similarly stained and used as positive and negative controls, respectively, for RSV immunostaining.
Viral Plaque Assays
At the end of the incubation period, the infected cells were harvested from each well by gentle scraping with an autoclaved rubber policeman, counted and placed in sterile eppendorf tubes before centrifugation at 500g for 5 min at 4°C in a microcentrifuge. The cells in the pellet were resuspended in 1 mL of cold MEM and sonicated on ice for 30 sec to release cell-associated virus. The amounts of released virus were quantified by plaque assay, with RSV-specific cytopathic effect (syncytia) further confirmed by immunostaining using anti-RSV antibody, as previously described. Results of plaque assays were expressed as numbers of plaque forming units (pfu) per 106 cells.
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