Mouse anti ssea1

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).

Immunofluorescence (IF) was performed after
fixation of the cultured cells in 4% paraformaldehyde
for 20 minutes followed by permeabilization with
0.2% Triton X-100 (Merk, USA) for 30 minutes.
The cells were subsequently blocked in phosphate-
buffered saline (PBS) supplemented with 10%
secondary antibodies host serum for 1 hour. The
blocked cells were incubated overnight at 4°C with
mouse anti-Oct4 (Santa Cruz, USA, sc5279), mouse
anti-SSEA-1 (R&D, MAB2155) and goat anti-Nanog
(Santa Cruz, USA, sc30329). The cells were washed
three times with PBS and subsequently incubated with
the following secondary antibodies goat anti-mouse
IgG-FITC (Santa Cruz, USA, sc2010), Alexa Fluor
568 goat anti-mouse (Invitrogen, USA, A21043),
and Alexa Fluor 568 donkey anti-goat (Invitrogen,
USA, A11057). The cells were stained with 1 µg/
ml DAPI for 10 minutes in the dark and after three
PBS washes, we used an Olympus fluorescent
microscope (Olympus, Japan) to visualize the cells.
Alkaline phosphatase (ALP) staining was performed
according to the manufacturer’s instructions using an
Alkaline Phosphatase Detection Kit.