Bone histomorphometric analyses were performed on uncalcified long bones and vertebrae in accordance with previously described methods (59 (link)). Briefly, the bone was fixed with 10% formalin, followed by dehydration in an ethanol series and subsequent embedding in methyl methacrylate resin, according to standard protocols. The BV/TV ratio was measured by von Kossa staining. Osteoblast and osteoclast parameters were analyzed by staining with toluidine blue and TRAP, respectively. Analyses were performed using the OsteoMeasure analysis system (OsteoMetrics), according to standard protocols (60 (link)). Trabecular architecture was assessed in long bones using a μCT system (Comscan), and the BV/TV ratio was measured using TRI/3D-BON software (RATOC) (61 (link), 62 (link)). Calcein, which has a high calcium affinity, was injected twice into mice with an interval of 3 days, and then, mice were killed 2 days after the last injection. The new-forming bone area was marked with the calcein-calcium complex. Concentrations of CTx and P1NP in serum were determined by enzyme-linked immunosorbent assay kits according to the manufacturer’s instructions (Immunodiagnostic Systems).
Enzyme linked immunosorbent assay kit
Manufactured by Immunodiagnostic Systems
Sourced in United Kingdom
An enzyme-linked immunosorbent assay (ELISA) kit is a laboratory tool used to detect and quantify specific proteins or molecules in a sample. The kit utilizes antibodies and color-changing enzymes to identify the presence and concentration of the target analyte.
Automatically generated - may contain errors
6 protocols using enzyme linked immunosorbent assay kit
1
Bone Histomorphometry and Microarchitecture Analysis
2
Serum Biomarkers of Bone Homeostasis
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Serum was collected via terminal bleeds from all mice. Soluble RANKL and OPG in blood plasma were measured using the mouse PicoKine Kit (BosterBio) according to the manual provided by the manufacturer. The serum levels of a bone formation marker, P1NP, and a bone resorption marker, CTX-1, were quantified using enzyme-linked immunosorbent assay kits from ImmunoDiagnostic Systems as described by the manufacturer.
3
Biochemical Assays in Clinical Research
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All biochemical tests were done by photometric assays (BT 2000) using Pars kit (Tehran, Iran). TPO-Ab, PTH, and TSH were measured by chemiluminescent immunoassay method (Advia Centaur CP, Siemens Healthcare Diagnostic Inc., USA).
Vitamin D measured by enzyme-linked immunosorbent assay kit (Immunodiagnostic Systems Limited, UK).
Vitamin D measured by enzyme-linked immunosorbent assay kit (Immunodiagnostic Systems Limited, UK).
4
Cytokine and Bone Marker Profiling
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Whole blood was obtained by cardiac puncture at time of harvest, allowed to clot at room temperature for ≥30 min, then centrifuged at 10,000g for 10 min. Systemic cytokines were assayed using a Meso Scale Discovery U-Plex electrochemiluminescence assay for a total of 20 cytokines according to manufacturer’s instructions at the Emory Multiplex Immunoassay core. The investigated markers were EPO, GM-CSF, IFN-γ, IL-1β, IL-4, IL-6, IL-10, IL-13, IL-16, IL-17a, IL-17c, IL-17e/IL-25, IL-17f, IL-21, IL-22, IL-23, IL-33, MCP-1, TNF-α, and VEGF-a. Undetectable concentrations of any cytokine were recorded as one-half of the lower limit of quantification, with the exception of GM-CSF, IL-13, IL-4, IL- 17c, IL-17e/IL-25, and IL-17f which were not detected in any sample and were excluded from analyses. N-terminal propeptide of type I procollagen (PINP) (ImmunoDiagnostic Systems) were assayed using an Enzyme-Linked Immunosorbent Assay kit according to the manufacturer’s recommendations.
5
Blood Serum Biomarker Analysis
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Blood was collected from the rats prior to euthanasia via the retro-orbital sinus. Serum was extracted immediately after centrifuging the blood at 3,000 rpm for 10 minutes. The serum was stored at −70°C until analysis. Osteocalcin and carboxy-terminal of type 1 collagen crosslinks were measured using an enzyme-linked immunosorbent assay kit (Immunodiagnostic Systems, Tyne and Wear, UK).
6
Serum Biomarkers for Bone Metabolism Assessment
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Samples were centrifuged, and the upper serum phases were collected and frozen at − 80 °C before the assays. Serum samples from participants were used to measure TP1NP, BAP, NTX, and intact parathyroid hormone (PTH int) levels. Serum levels of TP1NP were measured by electrochemiluminescence immunoassay using a Cobas e 411 analyzer (Roche Diagnostics K.K., Tokyo, Japan). The detectable range of the TP1NP assay was 5 to 1200 ng/ml. Serum BAP level was determined using an enzyme-linked immunosorbent assay kit (Immunodiagnostic Systems, Tyne & Wear, UK) with a detection sensitivity of 1.0 μg/l. PTH level was measured using an intact assay using a chemiluminescent method (Abbott i2000, TX, USA). The lower detection threshold of the PTH int assay was 1 pg/ml. Serum NTX levels were determined by a fully automated enzyme-linked immunosorbent assay (ELISA) using Osteomark NTx Serum ELISA Test Kits (Alere Inc., Seattle, WA).
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