Ab5922

Immunofluorescent staining was carried out to discriminate between tumor cells and fibroblasts using anti-fibroblast TE-7 (CBL271, Merck Millipore, Darmstadt, Germany), anti-nestin (AB5922, Merck Millipore) and secondary antibodies (ab6563, ab150081, abcam, Cambridge, UK). Briefly, for the detection of TE-7 the fixated co-cultures were permeabilized with 0.1% TritonX-100 at room temperature (RT) for 5 min. After blocking with 10% goat serum for 15 min, samples were incubated with anti-fibroblast TE-7 primary antibodies (dilution 1:100) at 5 °C overnight, washed with TBS (20 mM Tris, 134 mM NaCl) and subsequently incubated with the secondary antibody (1:250; ab6563) for 45 min at RT. For the detection of nestin, fixated cell cultures were permeabilized with 0.1% TritonX-100 in TBS for 1 h at RT, blocked for 15 min with 10% goat serum and then incubated with an anti-nestin antibody (dilution 1:250) for 1 h at RT. Afterwards, cultures were washed with TBS and incubated with a dilution of secondary antibody (1:250; ab150081) for 45 min at RT. Finally, nuclei were counterstained with DAPI (4 µg/ml) and cell cultures were preserved in 10% sodium azide solution at 4 °C until microscopy.

Cells were fixed in 4% paraformaldehyde for 30 min at room temperature, permeabilized with 0.2% TritonX-100 PBS and incubated with 1xBlock Ace (DS PHARMA BIOMEDICAL, Osaka, Japan) containing 0.2% Tween-20 to prevent non-specific binding before overnight incubation with primary antibodies at 4 °C. The following day, secondary antibody incubations were performed for 1 h with the appropriate species-specific antiserum coupled to either FITC or Cy3 (Jackson ImmunoResearch #711-095-152, #711-165-152, #715-095-151, #715-165-151, 1/100, West Grove, PA, USA). After staining the nuclei with DAPI (Sigma-Aldrich, 1/1,000, St. Louis, MO, USA), the cells were mounted using Vectashield (Vector Laboratories, Burlingame, CA, USA) and imaged using BZ-X710 (KEYENCE). All antibodies were diluted in 1xBlock Ace. The following primary antibodies were used at the indicated dilution rates: PAX6 (BD Biosciences #561462, 1/100, Franklin Lakes, NJ, USA), NESTIN (MERCK #AB5922, 1/1,000, Darmstadt, Germany), SSEA-4 (R&D SYSTEMS #MAB1435, 1/100, Minneapolis, MN, USA), OCT-3/4 (R&D SYSTEMS #AF1759, 1/100), PERICENTRIN (Abcam #ab4448, 1/1,000, Cambridge, UK), and α-Tubulin (Sigma-Aldrich # T9026, 1/1,000) N-CADHERIN (Abcam #18203, 1/1000). For immunostaining of a definitive endodermal marker, Alexa Fluor 488-conjugated SOX17 (BD Biosciences #562205, 1/100) was used without secondary antibody.