Tibiae were stripped of soft tissue, fixed in 4% PFA for 48 hours, decalcified in 10% EDTA for 3–4 weeks, and processed into paraffin as described previously followed by sectioning (5 μm) and staining for TRAP activity using the standard naphthol AS-BI phosphate post coupling method and counterstained with toluidine blue Pin et al. (2021) (link). Briefly, after equilibration in 0.2 M sodium acetate, 50 mM sodium tartrate, pH 5.0, for 20 min at RT, sections were incubated at 37°C in the same buffer containing 0.5 mg/ml naphthol AS-MX phosphate (Sigma Chem. Co., St. Louis, MO) and 1.1 mg/ml Fast Red Violet LB salt (Sigma) and counter- stained in toluidine blue. Images were taken at 5X and 40X using an Olympus BX51 fluorescent microscope and Olympus cellSense Entry 1.2(Build 7533) imaging software. TRAP-positive osteocytes and osteoclasts 1.5 mm distal from the growth plate were quantified using Osteomeasure software (OsteoMetrics.Inc) in a blind fashion. Toluidine blue-stained osteoblasts from the same sections were quantified 1.5 mm distal from the growth plate using the same software.
Cellsense entry 1.2 build 7533
Manufactured by Olympus
The CellSense Entry 1.2 (Build 7533) is a compact and versatile microscope system designed for cell culture and basic microscopy applications. It features a 10x eyepiece, 4x and 10x objective lenses, and a built-in LED illumination system. The CellSense Entry 1.2 is suitable for viewing and monitoring cell samples in a laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
Bx51 fluorescent microscope, by Olympus (3 mentions)
Osteomeasure high resolution digital video system software, by OsteoMetrics (2 mentions)
Naphthol as mx phosphate, by Merck Group (1 mentions)
Fast red violet lb salt, by Merck Group (1 mentions)
Osteomeasure software, by OsteoMetrics (1 mentions)
3 protocols using cellsense entry 1.2 build 7533
1
Histological Analysis of Murine Bone
2
Femoral Bone Histomorphometry Analysis
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Animals were injected intraperitoneally with 30 mg·kg−1 calcein and 50 mg·kg−1 alizarin 7 and 2 days before sacrifice, respectively, for dynamic histomorphometric measurements.32 (link) Femur were isolated at 2 and 9 months of age and fixed in 10% neutral buffered formalin and embedded in methyl-methacrylate using previously established methods. Histomorphometric analyses were performed using the Osteomeasure high resolution digital video system software (Osteometrics Inc, Decatur, GA, USA). Osteoblast and osteoclast parameters were scored in von-Kossa/McNeal- and TRAP/Toluidine blue-stained femoral bone sections, respectively. Dynamic measurements were performed on unstained femoral cortical and longitudinal sections. For the dynamic measurements in femoral cancellous bone, only five fields at the secondary spongiosa were scored. Images were taken using an Olympus BX51 fluorescent microscope and Olympus cellSense Entry 1.2(Build 7533) imaging software. The terminology and units follow the ASBMR Histomorphometry Nomenclature Committee guidance.47 (link) All histological procedures were performed at the Histology and Histomorphometry Laboratory (ACBP, ICMH, Indiana CTSI) at Indiana University School of Medicine.
3
Histomorphometric Analysis of Vertebral Bone
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Animals were injected intraperitoneally with 30 mg/kg calcein and 50 mg/kg alizarin 7 and 2 days before sacrifice, respectively, for dynamic histomorphometric measurements (Davis et al., 2018 (link)). Lumbar vertebrae (L1−L3) were isolated and fixed in 10% neutral buffered formalin and embedded in methyl-methacrylate. Histomorphometric analysis was performed using the Osteomeasure high resolution digital video system software (Osteometrics Inc., Decatur, GA, USA). Osteoblast and osteoclast parameters were scored in von-Kossa/McNeal- and TRAP/Toluidine blue-stained vertebral bone sections, respectively. Dynamic measurements were performed on unstained sections. Images were taken using an Olympus BX51 fluorescent microscope and Olympus cellSense Entry 1.2(Build 7533) imaging software. The terminology and units follow the ASBMR Histomorphometry Nomenclature Committee guidance (Dempster et al., 2013 (link)). All histological procedures were performed at the Histology and Histomorphometry Laboratory (ACBP, ICMH, Indiana CTSI) at Indiana University School of Medicine.
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