Mouse anti albumin

Cells were fixed with 4% paraformaldehyde for 15 min at room temperature, and then blocked with PBS containing 0.25% Triton X-100 (Sigma) and 3% BSA (Sigma) for 1 h at room temperature. The cells were then incubated with primary antibodies at 4 °C overnight, washed with PBS, and incubated with appropriate fluorescence-conjugated secondary antibody for 1 h at room temperature in the dark. Nuclei were stained with DAPI (Sigma). Primary and secondary antibodies were diluted in PBS containing 3% BSA. Images were captured at room temperature using an Olympus microscope (IX71) or a confocal microscope (TCS SP5; Leica) with a 63× oil objective. The p-ATM intensity was quantified by software LAS AF Lite (Leica). Antibodies used for immunofluorescence are as follows: rabbit anti-E-cadherin (Cell Signaling, 1:200), mouse anti-albumin (R&D, 1:200), mouse anti-p-ATM (Ser1981) (Thermo, 1:200), rabbit anti-HA-Tag (Cell Signaling, 1:200), rat anti-RPA32 (Cell Signaling, 1:200), rabbit anti-53BP1 (Novus, 1:200), goat anti-Nanog (R&D, 1:100), mouse anti-SSEA1 (R&D, 1:100), Cy3-conjugated donkey anti-mouse/rabbit/rat/goat IgG (Jackson Laboratories, 1:1 000), Cy5-conjugated goat anti-rabbit/mouse IgG (Jackson Laboratories, 1:1 000).

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).