Anti mouse igg horseradish peroxidase linked whole antibody

A549 subconfluent monolayers were infected with HRSV at a multiplicity of infection (MOI) of 3 plaque-forming units (pfu) per cell in DMEM with 2% FBS (DMEM2) and incubated for 90 min at 37 °C. After this time, the inoculum was removed, and fresh DMEM2 was added. Samples (culture supernatants and cell pellets) were collected at different times post-infection.
HRSV titers were determined in HEp-2 cell monolayers inoculated with serial dilutions of the culture supernatants for 90 min at 37 °C and then overlaid with 0.7% agarose in DMEM2. Five days post-infection (dpi), the cell monolayers were fixed with 4% formaldehyde and permeabilized with methanol. Plaques were visualized by one-hour incubation with a mixture of monoclonal antibodies previously obtained in our laboratory against the HRSV glycoprotein G, glycoprotein F, and phosphoprotein [29 (link)], followed by one-hour incubation with an anti-mouse IgG horseradish peroxidase linked whole antibody (Abcam, Cambridge, UK), and 3-amino-9-ethylcarbazole (AEC, Alfa Aesar, Ward Hill, MA, USA). Virus plaques, which were visible to the naked eye, were counted and HRSV titers calculated.

A549 were infected with HRSV at a multiplicity of infection (MOI) of 3 or 0.1 plaque-forming units (pfu) per cell in DMEM 2% FBS (DMEM2). Virus adsorption was allowed for 90 min at 37 °C. After this time, fresh DMEM2 was added. Culture supernatants and cell pellets were collected at different times post-infection. Virus titers were determined in the supernatants and total RNA and proteins were extracted from the cell pellets.
For HRSV titration, HEp-2 cell monolayers were incubated with serial dilutions of the infected cell supernatants for 90 min at 37 °C and then covered with 0.7% agarose (LM Sieve, Conda, Madrid, Spain) in DMEM2. Agarose was allowed to harden during 45 min at 4 °C, and then cells were transferred to a CO₂ incubator at 37 °C for five days. After this, cells were fixed with 4% formaldehyde in PBS for 45 min, which was followed by methanol permeabilization for 10 min. Virus plaques were visualized by one hour incubation with a mixture of monoclonal antibodies against HRSV [35 (link)], followed by one hour incubation with an anti-mouse IgG horseradish-peroxidase-linked whole antibody (Abcam, Cambridge, UK), and 3-amino-9-ethylcarbazole (AEC, Alfa Aesar, Ward Hill, MA, USA).