Sybr green 1

Markers for secretory oviduct epithelial cells (OVGP1, PAX-8, ESR1) and epithelial markers (Cytokeratin, ß-Catenin) were evaluated by immunofluorescence (IF). Cells were grown on glass cover slips, fixed with histofix 4% (Carl Roth, Karlsruhe, Germany) overnight at 4°C, and unspecific binding sites were blocked with either 5% BSA plus 10% goat serum (Abcam, Cambridge, UK) or Roti-ImmunoBlock (1:50, Carl Roth, Karlsruhe, Germany) for 1 h at room temperature. Cells were incubated with the primary antibody overnight at 4°C. Primary antibodies and their respective dilutions (in blocking buffer) are listed in Table 1. Corresponding secondary antibodies were purchased from Thermo Fisher Scientific (Dreieich, Germany) and diluted in PBS + 1% BSA: goat anti rabbit Alexa 647 (1:200, A21245), donkey anti goat Alex 568 (1:40, A11057), and goat anti mouse Alexa 488 (1:40, A11017). Incubation time was 1 h at room temperature. Nuclei were counterstained either with TO-PRO-3 iodide (Mobitec, Berkheim, Germany) or SYBR Green I (Mobitec, Berkheim, Germany). Images were captured by the confocal laser scanning microscope LSM 800 equipped with ZEN software (Carl Zeiss, Oberkochen, Germany).

Immunolocalization of marker proteins for cilia development (acetylated tubulin) and oviduct specific secretory activity (oviduct-specific glycoprotein) was conducted in morphologically differentiated cultures. Antigen retrieval was performed either enzymatically (acetylated tubulin) by incubation with 0.06% trypsin, pH 7.8 or via heat-induced antigen retrieval (oviduct-specific glycoprotein) using sodium citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0). Unspecific binding sites were blocked with 5% BSA and 10% goat serum in PBS (30 min, room temperature). Slides were incubated with the primary antibodies mouse monoclonal anti-human acetylated tubulin (Sigma T7451; 1:1000 in PBS with 1% BSA) or rabbit polyclonal anti-human oviduct-specific glycoprotein (Abcam ab118590; 1:500 in PBS with 1% BSA), respectively (overnight, 4 °C). The corresponding secondary antibodies were goat anti-mouse IgG, Alexa 568 (Invitrogen A-11031; 1:40 in PBS with 1% BSA) and goat anti-rabbit IgG, Alexa 647 (Invitrogen A-21245; 1:200 in PBS with 1% BSA), respectively, and were applied for 1 h at room temperature. Negative controls were performed by omitting the primary antibody. SYBR Green I (Mobitec, Berkheim) was used for nuclei counterstaining. Pictures were captured using a Zeiss LSM800 equipped with fluorescence optics and ZEN software.