An aliquot of RSV was 10-fold diluted in DMEM and 6-well plates seeded with Hep-2 cells were inoculated with each dilution and incubated for 2 hours at 37°C and 5% CO2. Four days after inoculation, the cells were fixed in 1% formaldehyde in phosphate-buffered saline (PBS) and plates were stored at 4°C overnight. The final titer of RSV was determined by immunostaining plaque assay using primary anti-RSV F monoclonal antibody (MAB 8599, EMD Millipore Corp., USA) and secondary horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG antibody (Southern Biotech) as described (Hwang et al., 2017 (link); Lee et al., 2017 (link)). The plaques were visualized using HRP-sensitive DAB colored substrate (Sigma-Aldrich). The RSV viral titer was expressed as plaque forming units (PFU) per ml.
Mab8599
Manufactured by Merck Group
Sourced in United States
MAB8599 is a laboratory equipment product. It is a monoclonal antibody that can be used for research and diagnostic purposes. The core function of this product is to specifically bind to a target antigen, allowing for detection or quantification of that target in various experimental or clinical settings.
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Lab products found in correlation
Horseradish peroxidase hrp conjugated goat anti mouse igg antibody, by Southern Biotech (1 mentions)
Alexa fluor 594 donkey anti rabbit igg antibody, by Thermo Fisher Scientific (1 mentions)
Fluoroshield with dapi histology mounting medium, by Merck Group (1 mentions)
Zen blue edition, by Zeiss (1 mentions)
Bp337 500, by Thermo Fisher Scientific (1 mentions)
Lsm 710 confocal laser scanning microscope, by Zeiss (1 mentions)
Alexa fluor 488 goat anti mouse igg antibody, by Thermo Fisher Scientific (1 mentions)
T9284, by Merck Group (1 mentions)
Secondary antibody against human igg, by Southern Biotech (1 mentions)
A0545, by Merck Group (1 mentions)
A21202, by Thermo Fisher Scientific (1 mentions)
A21448, by Thermo Fisher Scientific (1 mentions)
Ab150080, by Abcam (1 mentions)
Ab155539, by Abcam (1 mentions)
Ab150115, by Abcam (1 mentions)
5 protocols using mab8599
1
Immunostaining Plaque Assay for RSV
2
Quantifying RSV and MUC1 in Lung Spheroids
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After 3 and 7 days post-inoculation, spheroids were fixed with 4% paraformaldehyde (416785000 ACROS) for 15 min and permeabilized with 0.2% Triton X-100 (T9284-100 ml Sigma) in PBS for 20 min. Spheroids were blocked with blocking solution [1% BSA (A3912-100 g Sigma) and 0.1% Triton X-100 in PBS] for 1 h followed by overnight incubation with a mixture of mouse anti-RSV fusion protein antibody (MAB8599-Merck Millipore) at 1:200 and rabbit anti-human MUC1 antibody (12123-T24 Sino Biological) at 1:2500 in blocking buffer. The samples were then thoroughly washed with PBS/Tween (0.1%) (BP337-500-Fisher BioReagents) and incubated for 1 h with the corresponding fluorescent secondary antibodies (1:250) [Alexa fluor 488-goat anti-mouse IgG antibody (A11002-Life Technologies) and Alexa fluor 594-donkey anti-rabbit IgG antibody (A21207-Life Technologies)]. Finally, the immunostained spheroids were mounted with the Fluoroshield with DAPI histology mounting medium (F6057-Sigma), before being visualized by the LSM710 laser scanning confocal microscope (Zeiss). The fluorescence intensities of MUC1 and RSV-F captured on the images were quantified by employing ZEN blue edition (Zeiss) and presented as fold change values in infected spheroids as compared to their time matching controls.
3
RSV F Protein Antibody Characterization
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RSV F monoclonal antibody clone 131-2A (Millipore MAB8599) was used in RSV plaque assays. Murine monoclonal antibodies mAb1112 and mAb1243 (generous gifts of Dr. J. Beeler), and human mAb D25, mAb AM14, and mAb motavizumab (generous gifts of Dr. J. McLellan), were used to verify F protein conformation in ELISA analysis of VLPs and soluble F proteins, and for antibody blocking experiments. Palivizumab used for antibody blocking experiments was the generous gift of Dr. Jorge Blanco. The anti-RSV F protein HR2 antibody and anti-NDV F-tail antibody used for Western Blots are polyclonal antibodies specific to the HR2 domain of the RSV F protein or the cytoplasmic tail of NDV F protein [20 (link)]. The anti-RSV G protein antibody is a polyclonal antibody raised against a peptide containing G protein amino acids 180–198 (ThermoFisher Waltham, MA, USA). Secondary antibodies against goat, mouse, and rabbit IgG were purchased from Sigma. A secondary antibody against human IgG was purchased from Southern Biotech.
4
Monoclonal Antibodies for RSV Protein Analysis
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RSV F monoclonal antibody clone 131–2A (Millipore MAB8599) was used in RSV plaque assays. Monoclonal antibody (mAb) 1112, mAb 1200, mAb 1243, were generous gifts of Dr. J. Beeler37 (link) and used to verify F protein conformations, and mAb D25 and mAb motavizumab, generous gifts of Dr. J. McLellan,,14 (link) were used for ELISA analysis of VLPs and soluble F proteins. Anti-RSV F protein HR2 antibody used for Western Blots is a polyclonal antibody specific to the HR2 domain of the RSV F protein.18 (link) Anti-RSV G protein antibody is a polyclonal antibody raised against a peptide containing G protein amino acids 180–198 (ThermoFisher PA5–22827). Secondary antibodies against goat (A5420), mouse (A5906) and rabbit IgG (A0545) were purchased from Sigma.
5
Immunofluorescence Antibody Protocol
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The antibodies used for immunofluorescence (IF) were rabbit polyclonal anti-TGN46 (ABT95; Millipore), sheep polyclonal anti-TGN46 (Abd Serotec AHP500; Bio-Rad), rabbit polyclonal anti-SNX2 and anti-VPS26 (gifts from C. Haft, NIDDK, NIH) (43 (link)), Alexa Fluor 488-labeled mouse monoclonal antibody (MAb) anti-HRSV F protein (133-1H MAB8262X; Millipore), mouse MAb anti-HRSV N protein labeled with FITC (MAB858-3F N; Millipore), mouse polyclonal anti-HRSV M protein (44 (link)), rabbit polyclonal anti-giantin (PRB-114C-200; Covance), mouse MAb anti-RSV F protein (MAB8599; Millipore), rabbit polyclonal anti-Lamp-1 D2D11 XPR (9091T; Cell Signaling), mouse MAb anti-EEA1 (610457; BD), rabbit polyclonal HRS (ab155539), Alexa Fluor 594-labeled goat anti-rabbit (ab150080; Abcam), Alexa Fluor 647-labeled goat anti-mouse (ab150115; Abcam), Alexa Fluor 488-labeled donkey anti-mouse (A21202; Invitrogen), and Alexa Fluor 647-labeled donkey anti-sheep (A21448 Thermo Fisher).
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