594 conjugated secondary antibodies

Pancreatic tissues were fixed in 10% formalin, embedded in paraffin, cut into 4-μm-thick sections. Paraffin sections were deparaffinized in xylene, rehydrated by ethanol gradient, and then heated in a pressure cooker in a citrate-based antigen retrieval buffer. The sections were blocked and incubated with primary antibody α-F4/80 (1:200; Cat. No. ab100790, Abcam, Cambridge, MA) at 4 °C overnight. Subsequently, secondary antibody was added for 30 min, followed by DAB substrate buffer (Abcam, Cambridge, CA) for 5 min. Finally, the sections were counterstained with hematoxylin, dehydrated, and mounted under a coverslip. Immunofluorescence staining was performed according to conventional methods using the following antibodies: α-Amylase (1:200; Cat. No. ab21156, Abcam, Cambridge, MA), α-CK19 (1:200; Cat. No. TROMA-III, University of Iowa Hybridoma Bank), α-PCNA (1:50; Cat. No. sc-56, Santa Cruz Biotech, Santa Cruz, CA) and α-KRAS^G12D (1:1000; Cat. No. ab221163, Abcam, Cambridge, MA). AlexaFluro 488 (Cat. No. ab150157, Abcam, Cambridge, MA) and 594 conjugated secondary antibodies (Cat. No. ab150084, Abcam, Cambridge, MA) were used at a 1:200 dilution. The sections were mounted in DAPI containing media (Santa Cruz Biotech, Santa Cruz, CA), exposed to DAPI, FITC, and Texas Red filters, and images were superimposed.

HaCaT cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with a 0.5% (v/v) Triton X-100 in PBS for 15 min, and blocked in 5% BSA (Solarbio) for 1 h. Then, HaCaT cells were incubated in primary antibodies overnight at 4 ℃. Subsequently, the cells were incubated in Alexa Fluor 488 and/or 594-conjugated secondary antibodies (1:1000; Abcam) at room temperature for 2 h. Finally, DAPI staining solution (Beyotime) was employed to stain the nuclei of the cells. Images were collected by LSM980 confocal laser scanning microscopy (Carl Zeiss). The primary antibodies utilized in immunofluorescence (IF) staining included anti-Vim (1:400), E-cad (1:250), Ki67 (1:250), and p-Akt (1:250).

NF-κB nuclear translocation and HBeAg cellular localization were detected with monoclonal antibody to P65, and GST tag. For NF-κB nuclear translocation, Alexa Fluor® 488 or 594-conjugated secondary antibodies (Abcam, Cambridge, UK) were incubated for 2 h. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, Abcam, Cambridge, UK). Double immunofluorescence staining of α-SMA and F4/80 was conducted on liver cryosections and incubated overnight with primary rat anti-mouse F4/80 (Abcam, Cambridge, UK) and rabbit anti-mouse-α-SMA (Cell-Signaling Technology, Boston, USA) and then with secondary antibodies and DAPI. Immunolocalization of Desmin and ki67 was performed on liver cryosections and incubated with primary rat anti-mouse ki67 (Thermo Fisher Scientific, Waltham, MA) and rabbit anti-mouse-Desmin (Cell-Signaling Technology, Boston, USA). Images were captured using a fluorescence microscope (Olympus BX63, Tokyo, Japan) or confocal fluorescence microscopy (Leica TCS SP8).