Ab216484

Maxillary bones were excised and immediately fixed in 4% paraformaldehyde neutral buffer solution for 48 hours. Then, the maxillary specimens were decalcified with 10% EDTA at room temperature for 7 days until the alveolar bone could be easily penetrated followed by conventional dehydration and paraffin embedding. Immunohistochemical detection of C3 and RANKL as well as HE staining was performed in each group of samples. Examination and analysis were performed in blind. For immunohistochemical detection, 5 μm sections were prepared. Deparaffinized sections were treated with methanol containing 3% hydrogen peroxide before conducting antigen retrieval using a microwave oven at 95°C for 5 minutes, and cooling at room temperature for 15 minutes for two times. After washing with phosphate‐buffered saline (PBS), 5% bovine serum albumin was applied for 10 minutes. The sections were incubated with antibody 16‐18 hours overnight at 4°C. Then, after washing by PBS, a biotin‐conjugated secondary antibody was applied for 30 minutes. DAB chromogenic agent kit (Boster) was used to develop colour and the samples were counterstained with haematoxylin. Antibodies used were Rabbit Anti‐C3 antibody (1:200, Abcam, ab200999) and Rabbit Anti‐RANKL antibody (1:200, Abcam, ab216484).

Antigen retrieval was carried out in 10 mM of citric acid, pH 6.0, at 100 °C for one hour. Sections were incubated with primary antibody to IFT80 (PAB27850; Abnova, Taipei, Taiwan), acetylated α-tubulin (T6793; Sigma Aldrich, St. Louis, MO, USA), RANKL (ab216484; Abcam, Cambridge, MA, USA), and sclerostin (AF1589; R&D Systems, Minneapolis, MN, USA) overnight at 4 °C, as well as the matched negative control (I-1000 or I-5000; Vector Laboratories, Inc., Newark, CA, USA). HRP-conjugated secondary antibodies (705-035-147 or 111-035-144; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA), Alexa Fluor™ 647 tyramide reagent (B40958; ThermoFisher, Waltham, MA, USA), or Alexa Fluor™ 488 tyramide reagent (B40953) and DAPI mounting media (Sigma-Aldrich, St. Louis, MO, USA) were used. Four to six images per sample were taken at 40× objectives and assessed with NIS-Element software (Nikon) to examine the numbers of immunopositive osteocytes, divided by the area or total osteocytes on the compression side of the distobuccal root of maxillary 1st molar. Images were examined by a double-blinded examiner, and the results were confirmed with a second examiner. For all experiments, capture times were determined, so that the control IgG had no immunofluorescence.

Immunostaining of sections was investigated using RANKL (ab216484; Abcam, Cambridge, UK), OPG (ab203061; Abcam Cambridge, UK), Wnt3a (1H12L14, Thermo Fisher Scientific, Inc., Waltham, MA, USA), and Dkk1 (BS-2162R; Thermo Fisher Scientific, Inc., Waltham, MA, USA) antibodies and All-in-One Kit for Immunohistochemical Staining for Tissues (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Immunostained osteoblasts were counted using light microscope and image analyzer (Carl Zeiss, Oberkochen, Germany). 3 fields of each section and 3 sections were randomly selected for evaluation.