Anti alp

Bone samples were fixed with 4% paraformaldehyde, decalcified, dehydrated, embedded in paraffin, and cut into 5 μm slices, after which they were dewaxed by xylene and alcohol (in the following order: 100, 95, 75, and 50%). Slices were immersed in distilled water and HE staining was performed in accordance with standard protocols. Fields with positive staining were captured by microscopy. Additionally, primary antibody anti-ALP (abcam, UK) was incubated overnight at 4 °C and, subsequently, incubated with anti-mouse IgG-HRP (SV-0001; Boster) for 1 h. Then, sections were observed under the microscope (OPTEC CCD TP510) with a 100× objective and pictures were collected. The staining intensity was scored according to previous research (Lian et al. 2019 (link)) as follows: 0 (negative), 1 (weakly positive), 2 (moderately positive), and 3 (strongly positive). The percent of positivity was scored according to five categories: 0 (< 5%), 1 (5–25%), 2 (25–50%), 3 (50–75%), and 4 (> 75%). Finally, the percent positivity score was multiplied by the staining intensity score to generate final expression scores, which ranged from 0 to 12.

The protein concentration of each sample was measured using a QuantiPro BCA Assay Kit (KeyGen Biotech, Shanghai, China). The membranes were then incubated overnight at 4 ºC with specific anti-CD63 (diluted 1: 200; Abcam, Cambridge, MA, USA), anti-CD81 (diluted 1: 500; Abcam), anti-CD40 (diluted 1: 1000; Bioss Antibodies, Woburn, MA, USA), anti-ALIX (diluted 1: 1000; Abbexa Ltd., Cambridge, UK), anti-RUNX2 (diluted 1: 500, SAB, USA), anti-BMP-2 (diluted 1: 500, Bioworld Technology, St Louis Park, MN, USA USA), anti-ALP (diluted 1: 1000, Abcam), anti-GJA1 (diluted 1: 1000, Bioworld), anti-AP3B1 (diluted 1: 300, Proteintech, Rosemont, IL, USA) and anti-GAPDH (diluted 1: 5000, Bioworld). Incubation with the secondary antibody (diluted 1:500, ABclonal, Woburn, MA, USA) lasted 1 h. The ECL luminescent solution was configured to collect the blotting results with a Bio-Rad gel imaging system (Bio-Rad, Hercules, CA, USA), and the results were analyzed using Image Lab software.

The protein expression of ALP, IBSP and Sp7 were measured by using western blotting after osteogenic differentiation of hAVICs. The transfected hAVICs samples were fixed in 4% paraformaldehyde for 30 minutes, and then blocked with 0.2% Triton X‐100 and 3% goat serum in PBS. Cell lysate was separated by 12% sodium dodecyl sulphate‐polyacrylamide gelelectrophoresis (SDS‐PAGE) gel. Primary antibodies including anti‐Sp7 (Abcam, USA), anti‐ALP (Abcam), anti‐IBSP (Abcam) and anti‐GAPDH (Abcam) were incubated overnight at 4°C. After washing, membranes were incubated with secondary anti‐rabbit horseradish peroxidase‐conjugated antibodies (Elabscience Biotechnology Co., Ltd, China) for 2 hours at room temperature.

Total protein (50 μg) was separated in a Bis-Trispolyacryl amide gel and transferred onto a PVDF membrane (Millipore). The membrane was then incubated in 5% bovine serum albumin (BSA), and then incubated with a primaryantibody at 4°C overnight. Next, samples were incubated with IRDye800CWHRP-conjugated anti-IgG at room temperature for 1hour and visualized via chemiluminescence with an infrared laser scanning system (OdysseyLicor,Lincoln,NE,USA). The following primary rabbit-anti-human antibodies were used: anti-ANGPT2 (1:1000; Abcam); anti-Notch1 (1:500; Abcam); anti-Notch2 (1:500; Abcam); anti-Runx2 (1:1000;Abcam); anti-Sp7/Osterix (1:2000; Abcam); anti-ALP (1:2000; Abcam); anti-OCN (1:500; Abcam) and anti-GAPDH (1:2,500; Abcam).

DPCs were isolated by differential digestion as described above, after which total protein was extracted and normalized according to the manufacturer’s instructions. The primary antibodies were anti-APE1 (1:1,000; abcam, USA), anti-DMP1 (1:1,000; Santa cruz, USA), anti-DSP1-H (1:1,000; Santa cruz, USA), anti-OPN (1:1000; abcam, USA), anti-ALP (1:1,000; abcam, USA), anti-OSX (1:1,000; abcam, USA), anti-Axin (1:1000; abcam, USA), anti-Lef1 (1:1000; abcam, USA), anti-non-p (active) β-catenin (1:1000; CST, USA), anti-p-GSK-3β (1:1000; CST, USA), anti-P21 (1:1000; abcam, USA), anti-cyclin-D1 (1:1,000; Santa cruz, USA) and anti-GAPDH (1:10,000; Zen, China) used as internal control. Then, the membranes were rinsed with TBST (0.1% Tween-20 in 0.01 mol/L TBS), incubated with appropriate horseradish peroxidase conjugated secondary antibodies at 1:5000 (Santa Cruz, USA) at room temperature for additional 2 h, visualized by Image Quant LAS 4000 mini (GE, UK). Densitometry analysis on the bands was performed using the NIH image J software and normalizing the data to total protein levels (Supplementary Fig. 1. and Supplementary Fig. 2).

Whole cell lysates were extracted with lysis buffer (10 mM Tris-HCL, 1 mM EDTA, 1% SDS, 1% Nonidet P-40, 1∶100 proteinase inhibitor cocktail, 50 mM β-glycerophosphate, 50 mM sodium fluoride) for western blotting. The protein content of the lysate was determined using a protein assay kit (Beyotime Institute of Biotechnology) following the manufacturer's recommended protocol. The proteins were loaded on 10% SDS polyacrylamide gels, transferred to PVDF membranes (Millipore, Bedford, MA) and blocked with 5% nonfat milk powder in PBS with 0.1% Tween. The membranes were probed overnight with the following monoclonal primary antibodies: anti-ALP, anti-Col-1, anti-OCN, anti-eNOS and anti-P-eNOS (Abcam, Cambridge, UK) and anti-Akt and anti-P-Akt and anti-PI3K P85 (Cell Signaling Technology, Beverly, MA). The membranes were then incubated with antimouse horseradish peroxidase-conjugated secondary antibody (Boster, Wuhan, China). The blots were visualized using an enhanced chemiluminescence kit (Amersham Biosciences, Piscataway, NJ) according to the manufacturer's recommended instructions. The ratio of the targeted band to actin was considered to be the relative intensity of the targeted band.