After 2 days of culture, the cells were washed twice with PBS and fixed with 4% Paraformaldehyde (PFA, Sigma–Aldrich) for 10 min at room temperature followed by three PBS washes. Plates were then sealed with parafilm and stored in the fridge until immunofluorescence. Cells were permeabilized with PBS containing 0.1% Triton (Sigma–Aldrich; TPBS) for 10 min and washed in PBS three times before being incubated with a blocking solution containing 10% donkey serum (Merck) in TPBS for 30 min. The blocking solution was then aspirated and the samples were washed three times in TPBS washes. The cells were then incubated with osteocalcin (1:200, Abcam, Cambridge, UK) in 10% donkey serum and 0.1% TPBS overnight at 4 °C. After 24 h, the primary antibody solution was decanted and the cells were washed three times with PBS and incubated with the secondary antibody Alexa Fluor 488 (1:500, Abcam) in 5% donkey serum and 0.2% TPBS for 1.5 h at room temperature. Following this, cells were incubated for 5 min with Hoechst (1:5000, Life Technologies, Scoresby, Australia) and three PBS washes were performed prior to mounting the coverslips on a glass slide. Cells were then visualized under a fluorescence microscope (Inverted Laboratory Microscope, Leica DM IL LED, Wetzlar, Germany).
Osteocalcin
Manufactured by Abcam
Sourced in United Kingdom, United States
Osteocalcin is a protein found in bone and dentin. It is a marker of osteoblast activity and is often used to assess bone formation.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488, by Thermo Fisher Scientific (3 mentions)
β actin, by Merck Group (2 mentions)
Runx2, by Abcam (2 mentions)
C digit blot scanner, by LI COR (2 mentions)
Phosphorylated serine threonine kinase p akt, by Cell Signaling Technology (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
P erk, by Cell Signaling Technology (2 mentions)
Protease inhibitor, by Roche (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Donkey serum, by Merck Group (1 mentions)
Donkey serum, by Abcam (1 mentions)
Alexa fluor 488, by Abcam (1 mentions)
Hoechst, by Thermo Fisher Scientific (1 mentions)
Dm il led, by Leica camera (1 mentions)
Paraformaldehyde pfa, by Merck Group (1 mentions)
Paraformaldehyde solution, by Electron Microscopy Sciences (1 mentions)
Glycerol 2 phosphate, by Merck Group (1 mentions)
Dulbecco s phosphate buffered saline dpbs, by Thermo Fisher Scientific (1 mentions)
25 protocols using osteocalcin
1
Immunofluorescence Staining of Osteocalcin
2
Osteogenic Differentiation Assay
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We purchased Whatman 114 filter paper, glycerol-2-phosphate, ascorbic acid, alizarin red S and 4,6-diamidino-2-phenylindole (DAPI) from Sigma-Aldrich (St Louis, MO). We obtained the paraformaldehyde solution (16% (v/v)) from Electron Microscopy Sciences (Hatfield, PA). Mouse antibodies to Osteocalcin and Alexa 647-labeled antibodies to rabbit IgG were supplied by Abcam (Cambridge, MA). We purchased trypsin-EDTA, penicillin-streptomycin, phalloidin (Texas Red-X), fetal calf serum (FCS), fetal bovine serum (FBS), alpha-minimal essential medium (α-MEM) medium, and Dulbecco’s phosphate buffered saline (DPBS) from Invitrogen (Carlsbad, CA). We bought the OsteoImage kit from Lonza Walkersville Inc. (Walkersville, MD). We used all of the reagents as received without further purification.
3
Evaluation of hBMSCs Engineered with DEPTOR
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Eight-week-old male BALB/c nude (nu/nu) mice were obtained from Jackson Laboratory and housed in pathogen-free facilities under a 12-h light and 12-h dark cycle. hBMSCs stably infected with shDEPTOR #1, shDEPTOR #2, and shNC mixed with beta-tricalcium phosphate particles (SynthoGraft; Bicon, Boston, MA, USA) were transplanted subcutaneously under the dorsal space of the nude mice (N = 6 per group). The mice were sacrificed by CO2 asphyxiation 8 weeks after transplantation. The specimens were decalcified with 10% EDTA (pH 7.4), dehydrated, and then embedded in paraffin. Slices (5-μm thick) were prepared and stained with H&E and Masson’s trichrome. Immunohistochemical (IHC) staining using antibodies against Ocn (Osteocalcin, 1:200 dilution; Abcam, Cambridge, UK) and Deptor (1:100 dilution; Proteintech) was performed with a diaminobenzidine (DAB) Staining kit (ZSGB-BIO, Beijing, China) under the manufacturer’s instructions.
4
Immunohistochemical Analysis of Mouse Subcutaneous Tissues
5
Immunofluorescence Staining of Tissue Sections
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The tissue sections were dried at RT for 20 minute and then blocked with 1% BSA PBS for 3×10 minutes, the sections were incubated with primary antibody at 4 °C overnight. The sections were washed with 1% BSA PBS buffer and then incubated with the secondary antibody conjugated with Alexa-dye for 1 hour at RT in dark room. The sections were washed with 1% BSA PBS buffer and stained with 0.1% DAPI for 5 minutes at RT in dark room. The sections were then washed and mounted in anti-fade mounting media (Vector Laboratories). The stained slides were observed using an Olympus IX81 microscope or scanned using an iCys Research imaging cytometer (CompuCyte).
The following antibodies were used in this study: Col2 (1:100, Cat. ab34712, Abcam), osteocalcin (1:200, Cat. Ab14173, Abcam), Kitl (1:100, Cat. sc-13126, Santa Cruz), CXCL12 (1:100, Cat. sc-6193, Santa Cruz), α-SMA (1:100, Cat. ab5694, Abcam), CD146 (1:100, Cat. 134702, Biolegend), CD140a (1:100, Cat. 135902, Biolegend), Sca-1 (1:100, Cat. 108102, Biolegend), CD31 (1:150, Cat. 5550274, BD). The secondary antibodies conjugated with Alexa fluor 488 and Alexa fluor 555 were from Invitrogen. In study with mouse primary antibody, the M.O.M. immunodetection kit (Cat. BMK-2202, Vector Laboratories) was used to eliminate the endogenous mouse immunoglobulins background in the tissue.
The following antibodies were used in this study: Col2 (1:100, Cat. ab34712, Abcam), osteocalcin (1:200, Cat. Ab14173, Abcam), Kitl (1:100, Cat. sc-13126, Santa Cruz), CXCL12 (1:100, Cat. sc-6193, Santa Cruz), α-SMA (1:100, Cat. ab5694, Abcam), CD146 (1:100, Cat. 134702, Biolegend), CD140a (1:100, Cat. 135902, Biolegend), Sca-1 (1:100, Cat. 108102, Biolegend), CD31 (1:150, Cat. 5550274, BD). The secondary antibodies conjugated with Alexa fluor 488 and Alexa fluor 555 were from Invitrogen. In study with mouse primary antibody, the M.O.M. immunodetection kit (Cat. BMK-2202, Vector Laboratories) was used to eliminate the endogenous mouse immunoglobulins background in the tissue.
6
Western Blot Analysis of Osteogenic Markers
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Cells were lysed in a radio-immunoprecipitation assay buffer with protease inhibitors (PIA32963, Thermo Fisher Scientific) and phosphatase inhibitors (2,006,643, Calbiochem, Billerica, MA, USA). After cell lysis, proteins were fractionated by 10%–15% SDS gels and electro-transferred to polyvinylidene difluoride transfer membranes (IPVH00010, Millipore, Billerica, MA, USA). After blocking 1 h with a blocking buffer (1,706,404, Bio-Rad, Hercules, CA, USA), the membrane was incubated overnight with primary antibodies and then with secondary antibodies conjugated with horseradish peroxidase for 45 min (7074 S/7076 S, Cell Signaling, Danvers, MA, USA). We used antibodies against Lrp5, Runx 2, Snail, MSN, Calr, cleaved-caspase 3, caspase 3, CD91, p-CREB, and CREB (Cell Signaling), c-fos, NFATc1, Cathepsin K (Santa Cruz Biotechnology, Dallas, TX, USA), CD47 (Thermo Fisher Scientific), Collagen I (Novus Biologicals, CO, USA), Osteocalcin (abcam, Boston, MA, USA), and β-actin as a control (A5441, Sigma). The protein level was determined using a SuperSignal west femto maximum sensitivity substrate (PI34096, Thermo Fisher Scientific), and a luminescent image analyzer (LAS-3000, Fuji Film, Tokyo, Japan) was used to quantify signal intensities.41 (link) The levels of Calr and MSN in CW008-treated CM were determined using the ELISA kits (MBS263181 and MBS2709503; MyBioSource).
7
Comprehensive Protein Analysis Using Western Blot
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Western blot analysis was conducted using the procedure previously described 49 (link). We used antibodies against, β-catenin, Lrp5, Runx2, Snail, TGFβ, MSN, PDL1, CD44 (Cell Signaling, Danvers, MA, USA), MMP9, NFATc1, cathepsin K, OPN, alkaline phosphatase, FN1 (Santa Cruz Biotechnology), CD95, RANKL, OPG (Invitrogen, Carlsbad, CA, USA), Hsp90ab1, Osteocalcin (Abcam, Cambridge, UK), KDM3A (Proteintech, Rosemont, IL, USA), and β-actin (Sigma). The levels of Hsp90ab1 and MSN in Lrp5 CM were determined using ELISA kits (MBS7700502 and MBS2124159; MyBioSource). The level of PDL-1 in EO771 cells, which were treated with 0.05, 0.1, 0.5, and 1 μg/mL of TGFβ and Lrp5 CM, was determined using a cell ELISA kit (PI62200; Invitrogen).
8
Immunofluorescent Analysis of Fractured Radius
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Fractured radius specimens were prepared for immunofluorescent staining according to the standard protocol [23 ]. Briefly, the deparaffinized tissue sections in citrate buffer were heated at 95 °C for 10 min for antigen retrieval, followed by blocking for 1 h with 10% normal mouse serum. The primary antibodies were used against VEGF (dilution 1:50, Abcam, USA), MMP-9 (dilution 1:100, Abcam, USA) and osteocalcin (10 μg/mL, Abcam, USA) at 4 °C overnight. Afterwards, the sections were incubated with the goat anti-mouse secondary antibody conjugated with FITC for 1 h at the room temperature. Finally, all slides were stained with 4′,6-diamidino-2-phenylindole (DAPI). Images of immunofluorescent staining were collected through Pannoramic 250 Flash III, and analyzed by CaseViewer 2.3 and Image J software, respectively.
9
Quantitative Serum Biomarker Analysis
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Serum samples were collected for the quantitative detection of procollagen I N-terminal peptide (PINP) (abbexa, UK) and C-terminal telopeptides of type I collagen (CTX) (abbexa, UK), osteocalcin (Abcam, USA), and ALP (Abcam, USA) ELISA kits according to the instructions. Finally, we normalized the ELISA result using the concentration of total protein for each specimen.
10
Protein Expression in MC3T3-E1 Osteoblasts
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Total protein was extracted from MC3T3-E1 cells using RIPA lysis buffer (Beyotime Institute of Biotechnology). The protein concentration was determined using a BCA assay kit (Beyotime Institute of Biotechnology). A total of 30 µg protein/well was resolved using 10% SDS-PAGE and transferred to a PVDF membrane. Subsequently, 5% non-fat milk was used to block the membrane at 37˚C for 1 h, followed by incubation at room temperature for 1 h with primary antibodies as follows: Runt-related transcription factor 2 (RUNX2; 1:1,000; cat. no. 12556; Cell Signaling Technology, Inc.), collagen type I α 1 (COL1A1; 1:1,000; cat. no. ab34710; Abcam), osteopontin (1:1,000, cat. no. ab214050; Abcam), osteocalcin (1:1,000, cat. no. ab133612; Abcam), Tbx5 (cat. no. ab259980; Abcam) and GAPDH (1:2,500, cat. no. ab9485; Abcam). The membrane was then incubated with horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody (1:2,000, cat. no. #5127; Cell Signaling Technology, Inc.) at room temperature for 2 h. The bands were visualized by using an enhanced chemiluminescence (ECL) reagent kit (Shanghai Yeasen Biotechnology Co., Ltd.) and semi-quantified with Image J software (Version 1.49; National Institutes of Health).
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