Anti actin monoclonal antibody

Tissues were fixed in 4 % paraformaldehyde for 1 h, then in 30 % sucrose solution overnight for cryoprotection. 10–12 μm thick sections were cut using a Leica cryostat. Immunohistological detection of HBcAg was performed on frozen sections using primary rabbit polyclonal anti-HBcAg antibody (1:200; Abcam, England) and Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:200; Jackson ImmunoResearch, USA). Finally, the sections were stained with 4′,6-diamidino-2-phenylindole (DAPI) for nuclear indication. Immunofluorescence analyses were performed with a fluorescence microscope (Leica, German).
HBV core protein was also analyzed by a standard western blot procedure 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. The image was digitized using a scanner and signal was quantified using of Quantity One software (Bio-Rad).

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).

At 4 dpf, 30 larvae from the WT or il7r^−/− groups were harvested and lysed with lysis buffer containing RIPA (CWBiotech, Beijing, China), phenylmethanesulfonyl fluoride (PMSF, Sigma, St. Louis, MO, USA), Protease Inhibitor Cocktail (PI, Promega), Phosphatase Inhibitor Cocktail 2 (PPI2, Sigma) and Phosphatase Inhibitor Cocktail 3 (PPI3, Sigma) at a ratio of 100:1:1:1:1. Western blot analysis was performed as previously described^{11 (link)}. Anti-IL7R polyclonal antibody (1:400; Santa Cruz, sc-662, Dallas, TX, USA) was used as the primary antibody. Anti-actin monoclonal antibody (1:3000; Santa Cruz, sc-58679) was used as a loading control.