Approximately 5×103 cells per well were settled in a 24-well plate overnight and then transfected with 1 μg of plasmids per well. After incubation for 24 hours, the cells were fixed with 4% paraformaldehyde, treated with 0.5% Triton-X 100, blocked with 1% BSA at room temperature, and then incubated with specific primary antibodies against p65 (Santa Cruz, USA) or preS2 (Abcam, USA) at 4℃ overnight. Subsequently, the cells were incubated with an Alexa Fluor® 568 conjugated donkey anti-Rabbit IgG (H+L) secondary antibody or Alexa Fluor® 568 conjugate goat anti-Mouse IgG (H+L) secondary antibody (ThermoFisher Scientific, USA) for one hour and then incubated with DAPI for 5 minutes at room temperature. The expression of MHB and the subcellular localization of p65 were observed with a Zeiss confocal microscope (LSM 510 Meta, Germany). For the subcellular localization of MHBs, MHBs and ELP-1-CFP were cotransfected into SMMC-7721 cells and observed with a Nikon confocal microscope (A1R, Japan).
Alexa fluor 568 conjugated donkey anti rabbit igg h l secondary antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor® 568 conjugated donkey anti-Rabbit IgG (H+L) secondary antibody is a fluorescent-labeled secondary antibody used for the detection of rabbit primary antibodies in various immunoassay applications. It is conjugated with the Alexa Fluor® 568 fluorescent dye, which has excitation/emission maxima of approximately 578/603 nm.
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2 protocols using alexa fluor 568 conjugated donkey anti rabbit igg h l secondary antibody
1
Immunofluorescence Assay for MHB and p65
2
Immunofluorescence Analysis of Subcellular Protein Localization
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Approximately 5×10 3 cells per well were seeded in 24-well plate overnight, transfected with 1μg of plasmids, and incubated for 24 hours. Cells were fixed using 4% paraformaldehyde for 30 minutes, followed by incubation with 0.5% Triton-X 100 for 20 minutes at room temperature. After blocking with 1% BSA, cells were incubated with anti-p65 antibody (Santa Cruz ,USA) or anti-hepatitis B virus core antigen antibody (Abcam, USA) at 4℃ overnight, incubated with an Alexa Fluor ® 568 conjugated donkey anti-Rabbit IgG (H+L) secondary antibody or Alexa Fluor ® 568 conjugated donkey anti-Mouse IgG secondary antibody (ThermoFisher scientific, USA) for one hour at room temperature, and counterstained by DAPI for five minutes. Subcellular localization of the p65 subunit or HBcAg was examined under Zeiss confocal microscopy (LSM 510 Meta, Germany).
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