Cells grown on coverslips were washed with PBS, fixed with 3.7% formaldehyde in PBS for 15 min, and observed under a confocal laser microscope (FV1000 IX81; Olympus) using a 60× and 100× oil-immersion objective lens with a numerical aperture of 1.40. For immunostaining, fixed cells were permeabilized with 0.1% Triton X-100 or 50 μg/ml digitonin in PBS for 5 min, blocked with 10% newborn bovine serum in PBS for 30 min, and incubated with primary antibodies for 1 h. After washing, the cells were incubated with Alexa Fluor 647–conjugated goat anti-rabbit IgG secondary antibodies (Thermo Fisher Scientific) for 1 h.
Alexa fluor 647 conjugated goat anti rabbit igg secondary antibodies
Manufactured by Thermo Fisher Scientific
Alexa Fluor 647–conjugated goat anti-rabbit IgG secondary antibodies are fluorescently labeled antibodies that bind to rabbit primary antibodies. They are used in various immunoassay techniques to detect and visualize target proteins or other molecules.
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4 protocols using alexa fluor 647 conjugated goat anti rabbit igg secondary antibodies
1
Confocal Microscopy of Immunostained Cells
2
Immunostaining of Fluorescent Proteins
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Cells expressing fluorescent‐tagged protein were grown on coverslips and fixed in 3.7% formaldehyde in PBS for 15 min. For immunostaining, fixed cells were permeabilized with 50 µg/ml digitonin in PBS for 5 min, blocked with 10% NBS in PBS for 30 min, and incubated with primary antibodies for 1 h. After washing, the cells were incubated with Alexa Fluor 647–conjugated goat anti-rabbit IgG secondary antibodies (Thermo Fisher Scientific) for 1 h. The stained cells were observed under a confocal laser microscope (FV1000 IX81; Olympus) using a 100× oil immersion objective lens with an NA of 1.40. The images were acquired using FV10-ASW 2.1 imaging software.
3
Immunofluorescence Microscopy Protocol
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Cells were plated on coverslips and fixed in 3.7% formaldehyde in PBS for 15 min. For immunostaining, fixed cells were permeabilized with 50 µg/ml digitonin in PBS for 5 min, blocked with 10% newborn calf serum in PBS for 30 min, and incubated with primary antibodies for 1 h. After washing, cells were incubated with Alexa Fluor 647–conjugated goat anti-rabbit IgG secondary antibodies (Thermo Fisher Scientific) for 1 h. The stained cells were observed under a confocal laser microscope (FV1000 IX81; Olympus) using a 100× oil-immersion objective lens with NA of 1.40. The images were acquired using FV10-ASW 2.1 imaging software.
4
Immunostaining of Fixed Cells
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Cells were plated on coverslips and fixed in 3.7% formaldehyde in PBS for 15 min. For immunostaining, fixed cells were permeabilized with 50 μg/mL digitonin in PBS for 5 min, blocked with 10% newborn calf serum in PBS for 30 min, and incubated with primary antibodies for 1 h. After a washing step, the cells were incubated with Alexa Fluor 647-conjugated goat anti-rabbit IgG secondary antibodies (Thermo Fisher Scientific) for 1 h. The stained cells were observed under a confocal laser microscope (FV1000 IX81; Olympus) using a 100 × oil immersion objective lens with a numerical aperture of 1.40.
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