Vero cells in 6-well culture plates were infected with PEDVs at a multiplicity of infection (MOI) of 0.01. After 72 h, the cells were washed twice with PBS containing 0.05% Tween-20 (PBST), and the cells were fixed with cold formaldehyde and blocked with 1% bovine serum albumin (BSA). They were then incubated at 37°C for 2 h with 1: 2,000 diluted anti-PEDV S protein monoclonal antibody (Median, Chuncheon, South Korea). After a wash step with PBST, a 1:5,000 diluted Alexa Fluor 488 conjugated goat anti-mouse IgG (H+L) antibody (Abcam, UK) was added, and then the cells were further incubated at 37°C for 1 h. The cells were then incubated with 4′,6-diamidino-2-phenylindole (DAPI; Beyotime). Finally, the cells were washed, and then they were observed under a fluorescence microscope (Nikon, Japan).
Alexa fluor 488 conjugated goat anti mouse igg h l antibody
Manufactured by Abcam
Sourced in United Kingdom
Alexa Fluor 488 conjugated goat anti-mouse IgG (H+L) antibody is a secondary antibody that specifically binds to the heavy and light chains of mouse immunoglobulin G (IgG). It is conjugated with the Alexa Fluor 488 fluorescent dye, which can be detected using appropriate instrumentation.
Automatically generated - may contain errors
Lab products found in correlation
Fluorescence microscope, by Nikon (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
Podocin, by Abcam (1 mentions)
Alexa fluor 647 conjugated goat anti mouse igg h l antibody, by Abcam (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Diamidino 2 phenylindole dapi, by Solarbio (1 mentions)
Fluorescence microscope, by Zeiss (1 mentions)
Mouse anti cpv antibody, by Abcam (1 mentions)
8 well chambered slides, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade mountant with dapi, by Thermo Fisher Scientific (1 mentions)
Bz x800 fluorescence microscope, by Keyence (1 mentions)
4 protocols using alexa fluor 488 conjugated goat anti mouse igg h l antibody
1
Immunofluorescence Detection of PEDV S Protein
2
Immunofluorescence Staining of Kidney Tissues
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The frozen sections (3 μm) of the kidney tissues were fixed with 4% paraformaldehyde for 15 min at room temperature. Podocytes cultured on coverslips were fixed with cold methanol/acetone for 10 min at room temperature. Following blocking with 10% donkey serum for 60 min at room temperature, the slides were immunostained with primary antibodies against FN (cat. no. ab2413; Abcam), podocin (cat. no. SAB4200810; Sigma-Aldrich; Merck KGaA), synaptopodin (synap; cat. no. SAB3500585; Sigma-Aldrich; Merck KGaA), LC3 (cat. no. ab192890; Abcam), p62 (cat. no. MA5-27800; Invitrogen; Thermo Fisher Scientific, Inc.) at 4°C; subsequently, they were incubated with a secondary antibodies against Alexa Fluor 647-conjugated donkey anti-rabbit IgG H&L antibody (1:700; cat. no. ab150075; Abcam), Alexa Fluor 647-conjugated goat anti-mouse IgG H&L antibody (1:700; cat. no. ab150115; Abcam), Alexa Fluor® 488-conjugated goat anti-mouse IgG H&L antibody (1:500; cat. no. ab150113; Abcam) for 2 h at 37°C. The counterstaining of the cell nuclei was performed using 4′,6-diamidino-2-phenylindole (Sigma-Aldrich; Merck KGaA) for 10 min at room temperature. The images were obtained using a confocal microscope at a magnification of ×400.
3
Fluorescent Detection of FPV Infection
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CRFK cells infected with FPV isolate (MOI = 0.1) in 96-well plates were treated with cold 80% (v/v) acetone for 15 min, washed with PBS three times, exposed to a mouse anti-CPV antibody (Abcam, Cambridge, United Kingdom) at 37°C for 1 h, and then treated with an Alexa Fluor 488-conjugated goat anti-mouse IgG H&L antibody (Abcam, Cambridge, United Kingdom). Then, diamidino-2-phenylindole (DAPI) (Solarbio, Beijing, China) dye was added for fluorescent staining of cell nuclei. FPV infected cells were observed under a fluorescence microscope (ZEISS, Oberkochen, Germany) after being washed with PBS; cells evidencing intranuclear fluorescence were considered infected with FPV.
4
Immunocytochemistry of dsRNA in Cells
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For immunocytochemical analysis, the cells were cultured in 8-well chambered slides (Thermo Scientific, #154534) and treated as previously described. After three washes with PBS, cells were fixed with 4% formaldehyde for 20 min at RT. After two washes cells were incubated with a blocking-permeabilization buffer (5% goat serum and 0.3% Triton X-100 in PBS) for 1 h. Then cells were incubated with anti-dsRNA (J2) antibody diluted (2.5 μg/mL) in antibody diluent buffer (1% BSA in PBS) overnight at 4 °C in a humidified chamber. After three 5 min washes with PBS, the cells were incubated with Alexa Fluor 488 conjugated goat anti-mouse IgG H&L antibody (2 μg/mL) (abcam, #ab150113) at RT for 1 h in the dark. After three 5 min washes, the cells were mounted using the ProLong™ Gold Antifade Mountant with DAPI (Invitrogen, #P36935) and coverslips. Slides were imaged using a KEYENCE BZ-X800 fluorescence microscope.
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