Ab93876

Frontal sections (5 μm) of mouse heads were used for immunofluorescent detection. Histological sections were de-paraffinized in xylene and rehydrated in a gradient series of ethanol, finishing in water. Sections were pre-treated in citrate buffer (98°C/10 min) for antigen retrieval and then incubated with primary Anti-Caspase-12 antibody (2202, Cell Signaling, Danvers, MA) overnight. For immunofluorescence, cells grown on glass were fixed, pre-treated with 0.1% triton and incubated with the primary antibodies for caspase-12 (see above) or osteocalcin (ab93876, Abcam). Primary antibodies were followed by incubation with secondary anti-rabbit antibody Alexa Fluor 488 (Thermo Fisher Scientific, United States) (1:200) for 40 min at RT. Nuclei were detected by ProLong Gold Antifade reagent with DAPI (Thermo Fisher Scientific, United States). For immunohistochemistry, mandibular slices were treated as described above. Incubation with primary antibodies: osteocalcin, caspase-12 and Runx2 (sc10758, Santa Cruz Biotechnology) was followed by treatment with the peroxidase-conjugated streptavidin-biotin system (Vectastain) and the chromogen substrate diaminobenzidine (DAB, K3466; Dako). Slides were counterstained with hematoxylin. Negative and positive controls for Caspase-12 primary antibody are included in Supplementary Figure 1.

Total protein was isolated from tissues or cells (48 h after transfection) using radioimmunoprecipitation assay buffer. The protein was separated by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Afterward, proteins were transferred to a polyvinylidene fluoride (PVDF) membrane, and then blocked with 5% skim milk powder at room temperature for 1 h. Subsequently, the PVDF membrane was incubated with the corresponding diluted antibody overnight at 4°C. The following primary antibodies were used: rabbit antibodies to STC1 (1:1000, ab83065, Abcam, Cambridge, United Kingdom); p-JNK (1:1000, ab124956, Abcam); JNK (1:2000, ab208035, Abcam); bone morphogenetic protein-2 (BMP2; 1:1000, ab14933, Abcam). Osteocalcin (1:500, ab93876, Abcam). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH; ab9485, 1:2500) was used as a normalization medium. The blot was incubated with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (IgG) secondary antibody (ab97051, 1:2000, Abcam) for 1 h. Protein bands were visualized using the enhanced chemiluminescence detection kit (No. BB-3501, Amersham Pharmacia Biotech, Chicago, IL, United States) and Bio-Rad Image analysis system (Bio-Rad Laboratories, Inc., Hercules, CA, United States). Software Quantity One v4.6.2 was used for the further quantifying analysis.

To evaluate osteoblasts, immunostaining was performed using a standard protocol [17 (link)]. We incubated sections with primary antibodies to mouse Alkaline phosphatase (ALP, PA1004, Boster, Pleasanton, USA) and Osteocalcin (OCN, ab93876, Abcam, Cambridge, UK) overnight at 4°C. A biotinylated horseradish peroxidase detection system (Vectastain, PK-6200, Vector Laboratories, Burlingame, USA) was subsequently used to detect the immunoactivity, followed by incubation in 3,3'-diaminobenzidine (DAB, SK-4100, Vector Laboratories, Burlingame, USA) and counterstaining with hematoxylin. Also, tartrate-resistant acid phosphatase (TRAP) staining was performed for osteoclasts. Descriptive analysis to the immunostaining was performed by comparing the number of cells in the view field that are positive with the markers mentioned above. At least three mice per group were examined. Three equidistant sections spaced at 200 μm apart throughout the middle 1/3 coronal section of the vertebra were evaluated.

After micro-CT scanning, specimens were decalcified using 10% EDTA buffered with Tris-EDTA solution (pH 7.2–7.4, to 700 mL of PBS, add 100 g EDTA and adjust pH as needed to 7.2–7.4 by cautiously adding drops of 10 N NaOH) for 2 weeks. Before being embedded into a paraffin block, the plastic tube was removed. Those blocks were sectioned at a thickness of 4 μm and then stained with hematoxylin/eosin (HE) and Masson trichrome (MT). Histological images were acquired with a digital slide scanner (Paranoramic 250 Flash III, 3d-Histech, Budapest, Hungary). The acquired 3D images were measured using an image analysis program (Image Pro Plus, Media Cybernetics, Inc., Rockville, MD, USA). For immunohistochemistry, the sections were incubated overnight in the primary antibody at 4 °C. The next day, the sections were stained with Alexa Fluor^® 488 goat anti-rabbit IgG (H + L) (Invitrogen, Waltham, MA, USA, A10034) for 1 h at room temperature. Primary antibodies used in staining were rabbit-polyclonal to osteocalcin (mouse-specific, 1:100, Abcam, Cambridge, UK, ab93876), rabbit-polyclonal to osteopontin (mouse/human-specific, 1:100, Abcam, Cambridge, UK, ab8448), and rabbit-polyclonal to CD31 (human/mouse/pig-specific, 1:50, Abcam, Cambridge, UK, ab28364), which were used to demonstrate new bone formation and angiogenesis.