Mouse anti ck18

To analyze the expression of EMT markers in the HCC cells after SIRT1 downregulation, immunofluorescence staining was performed according to the manufacturer's protocol. Briefly, SIRT1-depleted cells were seeded onto coverslips and permeabilized with 0.25% Triton for 10 minutes. Nonspecific binding sites were blocked with 1% bovine serum albumin (BSA) for 30 minutes. Subsequently, the coverslips were stained with mouse anti-CK18 (Abcam, Cambridge, MA), rabbit anti-vimentin (Santa Cruz Biotechnology Inc.) and mouse anti-fibronectin (Santa Cruz) antibodies overnight, and then incubated with an Alexa Fluor 647-conjugated rabbit anti-mouse IgG or goat anti-rabbit IgG secondary antibody (Invitrogen, Carlsbad, CA). Nuclei were stained with DAPI, and the coverslips were mounted with an anti-fade reagent. Finally, the cells were analyzed under the confocal laser-scanning microscope.

Immunocytochemistry was performed as we described previously [35 (link)]. Briefly, cells were fixed with 4% paraformaldehyde (Sigma) for 20 min at room temperature and washed three times with PBS. Fixed cells were then permeabilized and blocked with DPBS (Welgene) containing 0.03% Triton X-100 (Sigma) and 5% FBS (Seradigm) for 1 hr at room temperature. Permeabilized cells were then incubated with a primary antibody mixture for 16 hrs at 4°C and incubated with the appropriate secondary antibody after washing three times. Counterstaining was performed with Hoechst 33342 (Sigma). Primary antibodies used for immunocytochemistry are as follows: mouse anti-albumin (R&D Systems, 1 : 100), rabbit anti-α-1-antitrypsin (Abcam, 1 : 100), mouse anti-CK18 (Abcam, 1 : 200), rabbit anti-E-cadherin (Cell Signaling, 1 : 200), and rabbit anti-ZO1 (Invitrogen, 1 : 200).

Cells were washed with PBS and fixed with 4% paraformaldehyde (Sigma) for 20 min at room temperature. The fixed cells were incubated with 0.3% Triton X-100 (Sigma) with 5% FBS in PBS for 2 h at room temperature. Cells were then incubated with the following primary antibodies overnight at 4 °C: mouse anti-albumin (R&D Systems), mouse anti-CK18 (Abcam), rabbit anti-ZO-1 (Invitrogen), rabbit anti-E-cadherin (Cell Signaling), mouse anti-vimentin (Abcam), mouse anti-Hnf4a (Abcam), goat anti-Hnf1a (Santa Cruz), rabbit anti-Foxa1 (Abcam), goat anti-Foxa3 (Santa Cruz), and rabbit anti-Gata4 (Millipore). After incubating with the primary antibody, the cells were washed three times with PBS and incubated with the appropriate fluorescently labeled Alexa-Fluor secondary antibody for 2 h at room temperature in the dark. Nuclei were counterstained with Hoechst33342 (Invitrogen).
Fluorescence-activated cell sorting (FACS) analysis was performed using a FACS Aria I (BD biosciences). Flow cytometry data were analyzed using the FlowJo software version 9.1 (Tree Star).