For further characterization, the cells were grown on sterile coverslips in 24-well plates. After 2 weeks, they were stained with antibodies to two different adherent junction proteins, i.e. e-cadherin, monoclonal IgG2α (BD Transduction Laboratories™, US) and ZO-1, polyclonal rabbit IgG (LSBio, Inc, US) and also to pan-cytokeratin, monoclonal IgG1 (Abcam, UK) to investigate their epithelial origin. Briefly, the cell medium was removed, then the cells were fixed in ice-cold acetone for 10 minutes, air dried and washed 3 times in PBS. Blocking was achieved with 5% skimmed milk in PBS for 30 min. The cells were then incubated with primary antibodies for 60 min. Alexa Fluor 488 conjugate (Invitrogen, US) were used as secondary antibodies, and cells incubated for 60 min. To visualize nuclei, the cells were incubated in propidium iodide for 5 min. The whole staining procedure was performed in the 24-well plates at room temperature. After staining, the cover slips were removed and mounted with SlowFade® (Invitrogen) on slides, and imaging was performed on a Zeiss LSM 710 confocal microscope, using 488 nm and 561 nm lasers.
Monoclonal igg1
Manufactured by Abcam
Sourced in United States
Monoclonal IgG1 is an immunoglobulin G1 (IgG1) antibody produced from a single clone of B cells. It is a type of antibody used in various laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Pore size centrifugal filters, by Merck Group (2 mentions)
Dabco, by Avantor (2 mentions)
Mowiol, by Avantor (2 mentions)
Poly l lysine (pll), by Merck Group (2 mentions)
Alexa fluor 488 conjugate, by Thermo Fisher Scientific (1 mentions)
Slowfade, by Thermo Fisher Scientific (1 mentions)
3 protocols using monoclonal igg1
1
Immunofluorescence Staining of Adherent Junctions
2
Capturing hRSV Filamentous Virions
3
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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