Rabbit polyclonal primary antibody to ghs r

For the detection of GHS-R, HGFs were grown on plastic coverslips (Thermo Fisher Scientific, Darmstadt, Germany) of 13 mm diameter in 24-well plates in the presence or absence of F. nucleatum for 1 d. After that, the cell monolayers were fixed in 4% paraformaldehyde (Sigma-Aldrich) at pH 7.4 and RT for 10 min. Subsequently, cells were permeabilized in 0.1% Triton X-100 (Sigma-Aldrich) for 5 min and then blocked using serum block (Dako) for 20 min. Afterwards, cells were incubated with rabbit polyclonal primary antibody to GHS-R (1:500, Abcam) for 90 min. Next, cells were incubated with goat anti-rabbit IgG HRP secondary antibody (Dako) for 45 min. Then, DAB solution, which was freshly prepared (3,3′-diaminobenzidine substrate diluted 1:10 in peroxidase substrate buffer), was added to the cells and left for 5–10 min at RT in dark. Cell monolayers were washed with PBS after each step. Cells were counterstained in Mayer’s hematoxylin solution for 5 s and washed thoroughly with water. Finally, HGFs were mounted with DePeX (SERVA Electrophoresis, Heidelberg, Germany). The slides were examined using an Axioskop 2 microscope (Zeiss, Oberkochen, Germany) equipped with a 20× objective. Images were captured using an AxioCam MRc microscope camera (Carl Zeiss) and the AxioVision 4.7 software (Carl Zeiss). Untreated cells served as control.

PDL cells were grown in the presence or absence of F. nucleatum on plastic coverslips (Thermo Fisher Scientific) of 13 mm diameter in 24-well plates for 1 d and 2 d. Cell monolayers were fixed in 4% paraformaldehyde (Sigma-Aldrich, Munich, Germany) at pH 7.4 and room temperature (RT) for 10 min and permeabilized in 0.1% Triton X-100 (Sigma-Aldrich) for 5 min followed by blocking using serum block (Dako, Hamburg, Germany) for 20 min. Afterwards, the cells were labeled with rabbit polyclonal primary antibody to GHS-R (Abcam, Cambridge, UK, 1 : 500) in a humid chamber at 4°C overnight and then incubated with goat anti-rabbit IgG HRP secondary antibody (Dako) for 45 min. The cells were rinsed with PBS (Invitrogen) in between each step. Finally, the cells were mounted with DePeX (SERVA Electrophoresis, Heidelberg, Germany) and the production of GHS-R was assessed with an Axioskop 2 microscope (20×, Carl Zeiss, Germany). The images were captured with an AxioCam MRc camera and analyzed with the AxioVision 4.7 software (Carl Zeiss). Untreated cells were used as a control.