Goat anti rabbit igg hrp secondary antibody

PDL cell stimulation with IL-1β (1 ng/mL) and F. nucleatum (OD: 0.025) was performed as described above, and cells were cultured on glass coverslips (Carl Roth, Karlsruhe, Germany) in 24-well plates for 24 h. Cells were prepared for immunocytochemistry by fixation in 4% paraformaldehyde (Sigma-Aldrich, Munich, Germany) at pH 7.4 and room temperature and by permeabilization with 0.1% Triton X-100 (Sigma-Aldrich). Each incubation step was followed by two washing steps with PBS (Sigma-Aldrich). To block unspecific background staining, cells were suspended in serum block (Dako, Hamburg, Germany) for 20 min, followed by incubation with rabbit polyclonal antibody anti-CTSS (Abcam, Cambridge, MA, USA; 1 : 250) at 4°C overnight. Cells were then labeled with a goat anti-rabbit IgG-HRP secondary antibody (Dako) for 45 min. Antibody binding was made visible by DAB chromogen (Thermo Fisher Scientific, Waltham, MA, USA) staining for 10 min at room temperature. Counterstaining with Mayer's hematoxylin (Merck, Darmstadt, Germany) for 1 min was followed, and finally, coverslips were mounted with DePex mounting medium (SERVA Electrophoresis, Heidelberg, Germany). An Axioskop 2 microscope (Carl Zeiss, Jena, Germany) with an AxioCam MRc camera (Carl Zeiss) and the AxioVision 4.7 software (Carl Zeiss) were used for standardized imaging.

After deparaffinization and dehydration, slides were treated by blocking endogenous peroxidase with 0.3% methanol (Merck)/30% H₂O₂ (Merck) for 10 min in the dark. For periostin staining, sections were pretreated with pepsin for 20 min at 37 °C. Subsequently, sections were pre-blocked with 1 × tris-buffered saline (Merck)/4% bovine serum albumin (Merck) for 1 h at room temperature and incubated in a humid chamber overnight at 4 °C with a rabbit polyclonal antibody against periostin with a concentration of 1:300 (ab14041, Abcam, Cambridge, UK). For the detection of osteopontin and osteocalcin, no pre-treatment of sections was needed. Sections were immediately incubated with a rabbit polyclonal antibody against osteopontin with a concentration of 1:300 for 1 h at room temperature (ab8448, Abcam) and with a mouse monoclonal antibody against osteocalcin with a concentration of 1:1,200 overnight in a humid chamber at 4 °C (ab13418, Abcam). Afterwards, sections were rinsed and incubated at room temperature with a goat anti-rabbit IgG-HRP secondary antibody or a goat anti-mouse IgG-HRP secondary antibody (Dako, Hamburg, Germany) for 30 min. Peroxidase activity was visualized by incubation with 3,3-diaminobenzidine chromogen (Thermo Fisher Scientific, Dreieich, Germany). Finally, sections were rinsed and counterstained for 30 s with Mayer’s hematoxylin (Merck).

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.