Ab65834

Macrophage lysates were prepared in lysis buffer (0.1% SDS, 25 mmol/L Tris-HCl pH 7.4, and protease inhibitors) and 40 μg of protein was separated by SDS-PAGE and blotted to polyvinylidenedifluoride membrane. Rabbit monoclonal anti-eNTPD1 (1/5000, ab108248, Abcam, Cambridge, United Kindom) and rabbit polyclonal anti-TNAP (1μg/mL, ab65834, Abcam) were used according to manufacturers’ instructions and as described[26 (link)].

Decalcification of the specimens was performed with 10% disodium ethylenediaminetetraacetic acid (EDTA-Na₂, Solarbio Beijing, China) at 4 °C for 30 d. Then, decalcified tissue embedded into paraffin wax was sliced into 5 μm sections. The sections were baked for 1 h at 60 °C and antigen repair was performed with trypsin antigen retrieval solution (BL333A, Biosharp, China). After blocking with goat serum for 2 h, the sections were incubated with primary antibodies overnight at 4 °C, then washed three times for 5 min. Finally, they were sealed with antifade mounting medium with DAPI (BL739A, Biosharp, China). In order to detect the bone regeneration, phenotype switching of macrophages, and angiogenesis in vivo, the tissue sections were incubated with primary antibodies anti-ALP (1:500, ab65834, Abcam), anti-Runx2 (1:500, ab76956, Abcam), anti-CD68 (1:250, ab955, Abcam)/anti-iNOS (1:200, ab15323, Abcam), anti-CD68/anti-CD206(1:200, ab64693, Abcam) and anti-CD31 (1:300, ab28364, Abcam). The fluorescent slides were finally covered by the cover glass using mounting medium containing DAPI. The fluorescent images were obtained with the fluorescence microscope, the fluorescence intensity of ALP and the number of Runx2⁺ cells, CD206⁺/CD68⁺ cells, iNOS⁺/CD68⁺ cells and CD31⁺ cells in the defects were counted and measured by Image J software.

Undifferentiated MC3T3E1 cranial bone progenitor cells, bone marrow stromal cells and Sol8 skeletal muscle progenitor cells were stained by immunofluorescence using Alexa Fluor 488 phalloidin for detection of F-actin (Thermo Fisher, Waltham, MA, USA), a rabbit anti-ALPL primary antibody (ab65834; Abcam, Cambridge, MA, USA), goat anti-rabbit Alexa Fluor-555 secondary antibody (Invitrogen, Carlsbad, CA, USA), and DAPI (4′,6-diamidino-2-phenylindole) nuclear stain to initially establish cellular localization of TNAP. For potential detection of co-localization of TNAP with mitochondria, cells were treated with 100 nM MitoTracker Red CMXRos (Invitrogen, Carlsbad, CA, USA) for 45 min. The mitochondrial dye was removed, and the cells were fixed. Cells were then stained with a monoclonal rat anti-ALPL primary antibody (MAB29091; R&D Systems, Minneapolis, MN, USA), donkey anti-rat Alexa Fluor-488 secondary antibody (Invitrogen, Carlsbad, CA, USA), and DAPI nuclear stain. Immunofluorescent staining was imaged using a Nikon Eclipse Ti microscope.

The assay represented the relative protein expression for osteogenic and odontogenic differentiation markers, miRNA’s target, and pathway protein. The total protein was isolated by RIPA lysis buffer (Beyotime, China) containing 1 mM PMSF (Beyotime, China). The protein sample was loaded onto 10% SDS-PAGE gel for electrophoresis, and then transferred onto 0.45 mm PVDF membranes (Millipore, USA) at 300 mA in a blotting apparatus (Tanon, China). Membranes were blocked with 2% non-fat milk for 2 h, then incubated in primary antibody overnight. The information of primary antibodies are as follows, RUNX2 (#12556, cell signaling technology, USA and ab23981, Abcam, UK), ALP (ab65834, Abcam, UK), OSX (ab22552, Abcam, UK), BMP2 (AF5163, Affinity, USA), BMP4 (BF0696, Affinity, USA and 12492-1-AP, proteintech, USA), LMBR1 (A18484, Abclonal, China), DSPP (BS71212, Bioworld, USA), GAPDH (10494-1-AP, proteintech, USA); Professor Jiang Hongbing of Jiangsu Province Key Laboratory of Oral Disease generously donated antibodies of CD63, CD9, and CALNEXIN. Next, the membranes were incubated in HRP-conjugated secondary antibody for 1 h, and signals were detected by enhanced chemiluminescence reagent (WBULS0100, Millipore, USA). Image J was used for the grayscale analysis of western blot.

MC3T3-E1 cells were cultured on the substrates a density of 4 × 10⁴ cells/cm² for 5, 7 and 14 days. Cells on each type of specimen were treated with cell lysis buffer (9803, Cell Signaling). The proteins were collected and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 5% Tris–HCl reducing gels, followed by transferring to the PVDF membrane (Millipore). The membrane was blocked by incubating in a blocking solution containing 5% skim milk (BD) and 0.1% TWEEN 20 (Fluka Chemika) for 2 h. After that, the sheets were incubated with ALP (ab65834, Abcam) or COL-I (ab6308, Abcam) at 4 °C overnight. Then HRP-anti-Rb antibody (050884, KPL) was used for the expression of ALP and COL-I. Band densities on the Western blots were assessed using the Quantity One software (Versa Doc 5000, BioRad) and normalized to GAPDH proteins.

Cryo-sections from cell implant studies were detected for tissue non-specific alkaline phosphatase (ALP; 1:300, ab65834, Abcam) and the nuclei were co-stained with DAPI. Imaging was performed using a Nikon Eclipse 90i Upright Fluorescence Microscope.