Cells were washed twice with PBS, fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.2% Triton X-100 in PBS for 15 min, and blocked with normal goat serum (Boster) for 1 hr at room temperature. Following, cells were incubated with HA tag antibody (1:500, Cell Signaling Technology) or pan-keratin antibody (1:500, Cell Signaling Technology). After 2 hr incubation, cells were washed for four times (5 min per washing) with PBST on table concentrator. Then secondary antibody conjugated 488 or 594 nm fluorophore (ThermoFisher) was incubated for 1 hr, and nuclei were stained with Hoechst 33342 (ThermoFisher). Images were captured with Leica confocal microscope.
Secondary antibody conjugated 488 or 594 nm fluorophore
Manufactured by Thermo Fisher Scientific
Secondary antibody conjugated to 488 or 594 nm fluorophore. This product is used to detect and visualize target proteins in various applications such as immunofluorescence, flow cytometry, and Western blotting.
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2 protocols using secondary antibody conjugated 488 or 594 nm fluorophore
1
Immunofluorescence Staining Protocol
2
Immunofluorescence Staining of Cells
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Cells were washed twice with PBS, fixed with 4% paraformaldehyde for 20 minutes, then permeabilized with 0.2% Triton X-100 in PBS for 10 minutes followed by incubation with normal goat serum (Boster, China) for 1 hr at room temperature. The cells were then incubated with a primary antibody for 2 hrs. After washing four times (5 min per time) with PBST (PBS containing 0.2% between 20), a secondary antibody conjugated 488 or 594 nm fluorophore (ThermoFisher) was added and incubated for 1 hr. The nuclei were stained with Hoechst 33342 (Thermo Fisher Scientific, USA). Images were captured with a Leica confocal microscope.
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