Anti ck19

Mouse eyeballs were excised and snap-frozen in Tissue-Tek OCT compound at 2 or 3 days after levobunolol, atenolol, or ICI 118, 551 treatment. The frozen sections (7 μm) were fixed in 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 for 15 min, and then blocked with 5% BSA for 1 h at room temperature. The sections were then incubated with anti-CK3 (Abcam, Cambridge, MA), anti-CK14 (Abcam), anti-CK19 (Proteintech, Chicago, IL), anti-Ki67 (Abcam), anti- phosphorylated epithelial growth factor receptor (pEGFR, Abcam), or anti-phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2, Cell Signaling Technology, Danvers, MA) overnight at 4 °C and subsequently incubated with fluorescein-conjugated secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h, followed by 4′, 6-diamidino-2-Phenylindole (DAPI, Solarbio, Beijing, China) staining for 5 min. All stainings were observed through an Eclipse TE2000-U microscope (Nikon, Tokyo, Japan) and were quantified by Image J software. Firstly, the images were put on, then, which were conversed to 8 bit and adjusted to gray scale followed by measuring parameters including the area, mean gray value (Mean), integral optical density. Finally, we got average gray scale of these images which were used to statistically analyze.

An equal amount of total protein was run on 10% SDS-PAGE (EpiZyme, cat. no. PG112), transferred to PVDF membranes (Merck millipore, cat. no. IPVH00010) (380 mA for 2 h), and probed with primary antibodies. The primary antibodies include anti-HBsAg (1:1000, 27 kDa, Novus Biologicals), anti-HBx (1:1000, 17 kDa, Abcam), anti-CK7 (1:1000, 51 kDa, Signalway antibody (SAB)), anti-CK19 (1:1000, 40 kDa, Signalway antibody (SAB)), anti-Hep-Par1 (1:1000, 165 kDa, Proteintech Group), anti-ALB (1:1000, 66 kDa, Proteintech Group), anti-α-Tubulin (1:1000, 55 kDa, Proteintech Group), anti-GAPDH (1:1000, 36 kDa, CST). The target protein bands were captured by binding of the secondary antibodies linked with peroxidase (1:1000, Anti-Rabbit IgG (H + L), CST) (1:1000, anti-mouse IgG (H + L), CST) to the primary antibodies.

Heat-induced epitope retrieval was used in liver paraffin sections. For cultured cells, 70% ethanol fixation and 4 N HCl permeabilization were used before staining. Immunofluorescence double staining were stained with anti-CK19 (Proteintech) and anti-BrdU antibody (Cell Signaling Technology, Beverly, MA, USA). Slides were visualized by Alexa Flour 488-labeled anti-mouse antibody and Alexa Flour 564-labeled anti-rabbit antibody (Invitrogen, Carlsbad, CA, USA).