Alexa fluor 488 conjugated goat anti rabbit igg

HBV core protein was analyzed by both immunofluorescence and western blot analysis. For immunofluorescence staining and confocal microscope analysis, the treated cells were seeded on coverslip and fixed with 4% paraformaldehyde. After being permeabilized with 0.1% (vol/vol) triton X-100 for 30 min, immunofluorescent detection of HBV core protein was performed on HepG2.2.15 cells using rabbit polyclonal IgG anti-human HBcAg primary antibody (1 : 200; Abcam, England) and Alexa Fluor 488-conjugated goat anti-rabbit IgG (1 : 200; Beyotime, China). Finally, the cells were stained with 4′,6-diamidino-2-phenylindole (DAPI, Vector, CA) for nuclear indication. Images were captured using a confocal laser scanning microscope (TCS-NT, Leica Microsystems, Heidelberg, Germany).
For western blot analysis, the treated cells were lysed in lysis buffer and cell lysates were separated in an SDS 12% polyacrylamide gel. The separated proteins were transferred to PVDF membrane and detected using primary polyclonal anti-HBcAg antibody (1 : 200; Abcam, England), anti-actin monoclonal antibody (1 : 1000; Santa Cruz Biotechnology, USA), and peroxidase-conjugated secondary antibodies followed by ECL detection. Immunoreactive bands were digitized using a scanner and signal was quantified using Image-Pro Plus (IPP) software (Version 6.0, Media Cybernetics, USA).

Cells were fixed in PBS containing 4% paraformaldehyde (NJ-reagent, Nanjing, China) for 30 min and permeabilized with phosphate-buffered saline containing 0.1% Triton X-100 (NJ-reagent) for 10 min. The samples were incubated with anti-CD133 antibody (1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA), followed by the secondary antibody conjugated to Cy3 NHS Ester goat anti-rabbit immunoglobulin (1:100; Beyotime, Shanghai, China).
The isolated CSCs was also stained with anti-ALDH antibody (1:100; Becton, Dickinson and Company, NJ, USA), followed by the secondary antibody conjugated to Alexa-Fluor 488-conjugated goat anti-rabbit IgG (1:200; Beyotime, Shanghai, China). Hoechst 33258 (Beyotime, Shanghai, China) was used for nuclear counterstaining.

PC-3 cells (5 × 10⁴ cells/well) were seeded in a 24-well chamber with a coverslip and incubated overnight. After treating with tryptamine (0.300 mM) for 8 h, the cells were washed with PBS and fixed with 4% paraformaldehyde at room temperature for 10 min, followed by 0.2% Triton X-100 for 5 min and blocked with 20% goat serum. The cells were incubated in rabbit anti-cleaved caspase-3 antibody (1:200, ab32042, Abcam) overnight at 4 °C, followed by incubation with secondary antibody Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:500, A0423, Beyotime, Shanghai, China) for 1 h at room temperature in the dark. Cells were washed and stained with 4′,6-diamidino-2-phenylindole (DAPI). Fluorescence images were acquired with a laser scanning confocal microscope (LSCM, Leica, TCS SP8 MP, Heidelberg, Germany).