To capture single hRSV filamentous virions on glass, hRSV A2 was propagated in HEp-2 cells at an MOI of 0.1. At 4 days post infection, the cell-associated and supernatant fractions were scraped, freeze-thawed and spun through 5 and 0.45 µm pore-size centrifugal filters (EMD Millipore) at 5000 × g and 4 °C for 4 and 1 min, respectively. The fraction between 0.45 and 5 µm in diameter was collected and immobilized onto a poly-l -lysine (Sigma Aldrich)-coated, first-surface mirror or cover glass by adsorption of 500 µl of filtered virus for 2 h at 4 °C. The immobilized virions were fixed using 4% paraformaldehyde and were immunofluorescently stained according to the aforementioned protocol. The antibodies used were anti-RSV F monocolonal (palivizumab, MedImmune, Gaithersburg, MD, USA) and anti-RSV N monoclonal (monoclonal IgG1, ab22501, Abcam). Coverslips were mounted in a mixture of Mowiol and DABCO (VWR)19 (link).
Dabco
Manufactured by Avantor
DABCO is a chemical compound used in various laboratory applications. It functions as a catalyst and can be used in a range of organic synthesis reactions.
Automatically generated - may contain errors
2 protocols using dabco
1
Capturing hRSV Filamentous Virions
2
Capturing hRSV Filamentous Virions
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To capture single hRSV filamentous virions on glass, hRSV A2 was propagated in HEp-2 cells at an MOI of 0.1. At 4 days post infection, the cell-associated and supernatant fractions were scraped, freeze-thawed and spun through 5 and 0.45 μm pore-size centrifugal filters (EMD Millipore) at 5000 × g and 4 °C for 4 and 1 min, respectively. The fraction between 0.45 and 5 μm in diameter was collected and immobilized onto a poly-l -lysine (Sigma Aldrich)-coated, first-surface mirror or cover glass by adsorption of 500 μl of filtered virus for 2 h at 4 °C. The immobilized virions were fixed using 4% paraformaldehyde and were immunofluorescently stained according to the aforementioned protocol. The antibodies used were anti-RSV F monocolonal (palivizumab, MedImmune, Gaithersburg, MD, USA) and anti-RSV N monoclonal (monoclonal IgG1, ab22501, Abcam). Coverslips were mounted in a mixture of Mowiol and DABCO (VWR)19 (link).
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