Vero cells were fixed with 4% paraformaldehyde for 20 min, washed with PBS, and then permeabilized with 0.1% Triton X-100 for 20 min at 37 °C. The treated cells were labeled with a mouse anti-PEDV mAb (monoclonal antibody) made in our laboratory (1:200 dilution) for 2 h at 37 °C. The cells were then washed three times with PBS and incubated with FITC-labeled goat anti-mouse IgG (H + L) antibody (1:200 dilution) (Beyotime, China) at 37 °C for 1 h. After being washed with PBS, the cells were visualized using confocal laser-scanning microscopy (Axiovert 200, Zeiss, Germany).
Fitc labeled goat anti mouse igg h l antibody
Manufactured by Beyotime
Sourced in China
FITC-labeled goat anti-mouse IgG (H + L) antibody is a secondary antibody used for immunological detection. It is produced by immunizing goats with mouse immunoglobulin and labeling the resulting antibodies with the fluorescent dye FITC (fluorescein isothiocyanate).
Automatically generated - may contain errors
4 protocols using fitc labeled goat anti mouse igg h l antibody
1
Immunofluorescence Staining of PEDV in Vero Cells
2
Immunofluorescence Assay for HA-Tagged Proteins
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DF-1 cells cultured in 35 mm dishes were transfected with the plasmids expressing HA tag, HA-M, or HA-M/3A protein. At 24 hpt, cells were rinsed thrice with PBS, fixed with 4% paraformaldehyde for 15 min, and then permeabilized with 0.25% Triton X-100 for 5 min. Cells were rinsed thrice with PBS and blocked with 10% FBS in PBS for 30 min at 37 °C, and then incubated with mouse anti-HA antibody diluted in PBS containing 10% FBS for 1 h at 37 °C. After three washes with PBS, the cells were incubated with FITC-labeled goat anti-mouse IgG(H+L) antibody (Beyotime Biotechnology, Shanghai, China) for 1 h at 37 °C. Cells were counterstained with DAPI (Sigma, USA) to detect the nuclei. Images were captured with an inverted fluorescence microscope and then dealt with Adobe Photoshop 8.0 software.
3
Tomatidine Inhibits PEDV Infection in Vero Cells
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An IFA was used to examine the effect of tomatidine on PEDV infection in Vero cells. Serially diluted tomatidine was added to the culture medium of the cells (final concentrations were 0.5, 2, 4, 6, 8, and 10 μM). DMSO was used as the negative control. PEDV (0.01 MOI) was then added to the cells, and cultures were incubated for 16 h at 37 °C. The cells were then fixed with 4% paraformaldehyde for 20 min, washed with PBS, and then permeabilized with 0.1% Trition X-100 for 20 min at 37 °C. The treated cells were incubated with a mouse anti-PEDV N-protein mAb prepared in our laboratory (1:200 dilution) for 2 h at 37 °C. The cells were then washed three times with PBS and incubated with FITC-labeled goat anti-mouse IgG (H + L) antibody (1:200 dilution) (A0568, Beyotime, China) at 37 °C for 1 h. After washing with PBS, the cells were visualized using a Zeiss inverted fluorescence microscope. The integrated optical density of fluorescence was determined using ImageJ software.
4
Immunofluorescence Assay for Porcine Viral Isolates
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To study the reactivity of McAbs with different isolates, the IFA was used previously described, with some modification (Zhang et al., 2019 (link)). Briefly, Vero cells were inoculated with PEDV strains CV777, YZ, SH, MS, respectively and ST were inoculated with TGEV. After absorption for 1 h, the viruses were discarded and DMEM containing 8 μg/ml trypsin (Boisharp, China) was added. When cytopathic effect (CPE) were observed, the cells were gently washed with PBS and fixed with absolute ethyl alcohol for 30 min at 4 °C. The plates were incubated with 2% BSA for 2 h at 37 °C after washing three times. Subsequently, McAbs (1:200 dilution) were added to the cell plates and acted 1 h at 37 °C after washing three times with PBS. A secondary antibody, FITC-labeled Goat Anti-Mouse IgG (H + L) antibody (1:100 dilution) (Beyotime, China), was added to wells for 45 min at 37 °C. Finally, the cells were washed three times in PBS and evaluated by inverted fluorescence microscope. The positive serum against PEDV and TGEV were used as control. RPMI-1640 containing 20 % FCS were used to incubate the control cells to eliminate the effect of medium on McAbs.
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