Tissue sections (n=20 in non-diabetic and diabetes groups, respectively; specimens were randomly selected; n=20 in control group) from lateral tibial plateau were evaluated using immunohistochemistry as described previously.14 (link),17 (link) In brief, biomarker of the osteoclasts (tartrate-resistant acidic phosphatase, TRAP) was detected using TRAP staining with a commercial TRAP kit (Sigma-Aldrich, Missouri, USA). To detect biomarkers of osteoblasts (osteocalcin) and osteoprogenitors (osterix),14 (link),17 (link) sections underwent heat-induced antigen retrieval in citrate buffer, followed by incubation with either anti-osterix (Abcam, Cambridge, UK) or anti-osteocalcin (TakaRa, Shiga, Japan) primary antibodies overnight. Next, horseradish peroxidase-labeled secondary antibodies (Abcam) was added and incubated for 60 min. Color was developed using diaminobenzidine (DAB) as substrate (Vector Lab, California, USA). After images were captured, the number of positive stained cells was quantified as previously described.14 (link),17 (link) Briefly, five sequential sections from each sample were stained and for each section, five areas were measured.14 (link),17 (link)
Anti osteocalcin
Manufactured by Takara Bio
Sourced in United Kingdom, Germany
Anti-osteocalcin is a laboratory reagent used for the detection and quantification of osteocalcin, a protein found in bone and dentin. It can be used in various assays and research applications to measure osteocalcin levels.
Automatically generated - may contain errors
Lab products found in correlation
Anti osterix, by Abcam (3 mentions)
Horseradish peroxidase labeled secondary antibody, by Abcam (2 mentions)
Trap kit, by Merck Group (1 mentions)
Diaminobenzidine dab, by Vector Laboratories (1 mentions)
Envision system, by Agilent Technologies (1 mentions)
Anti axin2, by Abcam (1 mentions)
Apoptag peroxidase in situ apoptosis detection kit, by Merck Group (1 mentions)
Anti gfp, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594 labeled anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Human pth 1 34, by Bachem (1 mentions)
Anti sox9, by Abcam (1 mentions)
5 protocols using anti osteocalcin
1
Quantifying Osteoblast and Osteoclast Markers in Diabetes
2
Osteoblast Differentiation and Matrix Characterization
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The differentiation of the osteoblasts was characterized by determination of the synthesis of bone matrix proteins. Cells were seeded at a concentration of 60 000 cells per cm2 and cultivated for 4 weeks. For immunohistochemistry, the cell culture medium was decanted. The specimens were washed three times with phosphate-buffered saline (PBS). Specific antibodies were used to detect extracellular matrix proteins by immunohistochemical staining.
Anti-collagen type I, polyclonal was obtained from BioTrend Chemikalien GmbH, Germany, the antibodies anti-osteocalcin and anti-osteonectin were obtained from Takara, Shiga, Japan and anti-osteopontin from CHEMICON International Inc., Temecula, USA. For immunohistochemical staining, the DAKO EnVisionTM + −system was applied. The stained cell cultures were controlled and analysed by light microscopy. Richardson staining was accomplished with a blue dye (Methylen blue Azur II).
Anti-collagen type I, polyclonal was obtained from BioTrend Chemikalien GmbH, Germany, the antibodies anti-osteocalcin and anti-osteonectin were obtained from Takara, Shiga, Japan and anti-osteopontin from CHEMICON International Inc., Temecula, USA. For immunohistochemical staining, the DAKO EnVisionTM + −system was applied. The stained cell cultures were controlled and analysed by light microscopy. Richardson staining was accomplished with a blue dye (Methylen blue Azur II).
3
Histological Analysis of Mouse Skeletal Tissues
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Postnatal and adult mouse femurs were dissected and fixed in 4% PFA/PBS for 2 days at 4 °C. Bone tissues were decalcified in 20%EDTA for 5days. Embryonic and neonatal tissues were fixed overnight in 4%PFA/PBS, but not decalcified. Fixed skeletal tissues were dehydrated through ethanol and embedded in paraffin; 8-μm serial sections were then prepared for histological analysis49 (link). Hematoxylin and Eosin (HE) staining and immunohistochemical analyses were carried out by standard procedures using the following antibodies (Ab)50 (link): anti-Cathepsin K (1:300, cat. no. ab19027, abcam)51 (link), anti-Osterix (1:1000, cat. no. ab22552, abcam)52 (link), anti-Axin2 (1:300, cat. no. ab32197, abcam)53 (link), anti-Osteocalcin (1:1000, cat. no. M173, Takara)54 (link), anti-Alkaline Phosphatase (1:500, cat. no. M190, Takara)55 (link). Bone-tissue cells with positive immunohistochemical signals representing equal areas of control and sFRP4 KO mice were counted and their average numbers were compared.
4
Immunostaining and PTH Administration for Bone Analysis
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For immunostaining of histological sections, mice were perfused with 4% paraformaldehyde followed by PBS. Decalcified tibiae were embedded in paraffin and the sections were prepared. Apoptotic cells were detected using the ApopTag Peroxidase in Situ Apoptosis Detection Kit (Merck Millipore). Methyl green was used for nuclear counterstaining. For immunofluorescent staining, mice were perfused with 4% paraformaldehyde followed by PBS. Femur were further fixed with 4% PFA for 1hr at 4 C. They were equilibrated with 30% sucrose in PBS at 4 C over night and embedded in SCEM (Leica Microsystems). Frozen sections were prepared as described previously (Kawamoto and Kawamoto, 2014) . The sections were subjected to immunostaining with anti-osteocalcin and anti-GFP antibodies to identify osteoblasts and IL-7-GFP, respectively. Antibodies used in this study were: anti-osteocalcin (Takara Bio Inc.), anti-GFP (catalog no. A10262, Life technologies), Alexa Fluor 488labeled anti-chicken IgY (A11039) and Alexa Fluor 594-labeled anti-rabbit IgG (A11012) antibodies.
In Vivo PTH Administration Human PTH (1-34) (Bachem) (40 mg per kg body weight) or saline was administered intraperitoneally once daily for 2 weeks before cecal ligation and puncture.
In Vivo PTH Administration Human PTH (1-34) (Bachem) (40 mg per kg body weight) or saline was administered intraperitoneally once daily for 2 weeks before cecal ligation and puncture.
5
Quantifying Bone Cell Biomarkers via IHC
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Biomarkers of osteoclast (tartrate-resistant acidic phosphatase, TRAP), osteoblast (Osteocalcin), osteoprogenitor (Osterix) 3, 4 , and biomarkers of the chondrogenic phase during endochondral bone formation (SOX9) 30 were detected using immunohistochemistry with protocols described previously (n ¼ 15; samples were randomly selected from each group) 3, 4, 31 . TRAP staining was performed according to the manufacturer's protocol (Sigma). Other sections were incubated overnight with either anti-Osterix (Abcam), anti-Osteocalcin (TakaRa), or anti-SOX9 (Abcam) primary antibodies, followed by horseradish peroxidase-labeled secondary antibodies (Abcam) and developed with 3, 30diaminobenzidine substrate (Vector Laboratories). After image capture, the number of positive cells was quantified as previously described by a blinder (MG) 3, 4 .
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