Dapi 4 6 diamidino 2 phenylindole dihydrochloride

Indirect immunofluorescence was performed as reported [25 (link)]. Following primary antibodies were used: rabbit polyclonal antibodies against pan-cadherin (ab16505; Abcam, Cambridge, UK) and against β-hCG (SAB4500168; Sigma-Aldrich, Taufkirchen, Germany), and mouse monoclonal antibody against acetylated α-tubulin (6-11B1, T7451; Sigma-Aldrich, Taufkirchen, Germany). Cy3-conjugated secondary antibodies were obtained from Jackson Immunoresearch (Cambridgeshire, UK). DAPI (4’,6-diamidino-2-phenylindole-dihydrochloride; Roche, Mannheim, Germany) was used to stain the DNA content. All slides were examined with an AxioObserver.Z1 microscope (Zeiss, Göttingen, Germany) equipped with an AxioCam MRm camera (Zeiss, Göttingen, Germany). The immunofluorescence stained slides were further imaged with an confocal laser scanning microscopy (CLSM) as described [30 (link)].

For indirect immunofluorescence staining, the adherently growing HUVEC, MSC, and pOB were cultured in 8-well chamber slides (BD BioCoat^TM culture slides) until 70–80% confluence, whereas the lineage-negative CBMNC were attached on culture slides by cytospin centrifugation. The cells were fixed on the culture slides by ice-cold methanol for 10 min at −20°C, washed twice with PBS for 5 min and used immediately for the staining procedure. The primary antibodies were diluted in 0.1% BSA/PBS and incubated for at least 1 h in a dark humid chamber. After washing, the fluorochrome-conjugated secondary antibodies diluted in 0.1% BSA/PBS was added and incubated for 1 h. For counterstaining, the secondary antibodies were combined with 1 μg/ml DAPI (4′,6-diamidino-2-phenylindole dihydrochloride; Roche, Mannheim Germany). The cells were washed three times with PBS and embedded in mounting medium (Dako fluorescence mounting medium; Dako, Hamburg). Control staining was performed by omitting the primary antibody. The labeled cells were examined using the fluorescence microscope Zeiss Axiophot (Göttingen, Germany).

For indirect immunofluorescence staining, cells were seeded on Nunc™ Lab-Tek™ II CC2™ chamber slides from Thermo Fisher Scientific (Schwerte). Briefly, cells were fixed for 15 min with 4% PFA and permeabilized for 5 min with 0.1% Triton™ X-100 at room temperature. The following primary antibodies were used for staining: polyclonal rabbit antibody against pericentrin (abcam^®, Cambridge, UK), monoclonal mouse antibody against FITC-conjugated α-tubulin (Sigma-Aldrich) and human immune serum against centromere (anti-centromere antibody, ACA, ImmunoVision, Springdale, USA). Cy3 and Cy5-conjugated secondary antibodies were obtained from Jackson Immunoresearch (Newmarket, UK). DNA was stained using DAPI (4′,6-diamidino-2-phenylindole-dihydrochloride, Roche). Slides were examined using an AxioObserver.Z1 microscope with a HCX PL APO CS 63.0×1.4 oil UV objective (Zeiss, Göttingen) and images were taken using a confocal laser scanning microscope (CLSM, Leica CTR 6500, Heidelberg).