Download Bone Research Protocols [Methods in Molec Bio 0816] 2nd ed. - M. Helfrich, et. al., (Humana, 2012) WW) PDF

TitleBone Research Protocols [Methods in Molec Bio 0816] 2nd ed. - M. Helfrich, et. al., (Humana, 2012) WW)
TagsMedical
LanguageEnglish
File Size20.0 MB
Total Pages657
Table of Contents
                            MMB_v816_9781617794148_1617794147
front-matter
	Bone Research Protocols
		Preface
		Contents
		Contributors
Part 1 Culture of osteoblasts and osteocytes
	Ch01_Primary Human Osteoblast Cultures
		Chapter 1: Primary Human Osteoblast Cultures
			1. Introduction
				1.1. Human Primary Osteoblast Cultures
				1.2. Osteosarcoma Cell Lines
				1.3. Osteoblasts from MSCs
				1.4. Human Osteoblasts from Peripheral Blood
			2. Materials
				2.1. Equipment
				2.2. Cell Isolation and Culture
				2.3. Phenotypic Characterisation
			3. Methods
				3.1. Establishing Primary Explant Cultures
				3.2. Secondary Explant Cultures
				3.3. Passaging Explant Cultures
				3.4. Ascorbate Containing Cultures
				3.5. Mineralising Cultures
				3.6. Cryopreservation
				3.7. Retrieval of Cryopreserved Cells
			4. Notes
			References
	Ch02_Osteoblast Isolation from Murine Calvaria and Long Bones
		Chapter 2: Osteoblast Isolation from Murine Calvaria and Long Bones
			1. Introduction
			2. Materials
				2.1. Tissues
				2.2. Instruments
				2.3. Media and Solutions
			3. Methods
				3.1. Isolation and Culture of Primary Bone Cells from Adult Mouse Long Bones
				3.2. Isolation and Culture of Primary Bone Cells from Adult Mouse Calvaria
				3.3. Isolation and Culture of Bone Cells from Neonatal Mouse Calvaria
				3.4. Isolation and Culture of Bone Cells from Neonatal Mouse Calvaria (Alternative)
			4. Notes
			References
	Ch03_Rat Osteoblast Cultures
		Chapter 3: Rat Osteoblast Cultures
			1. Introduction
			2. Materials
			3. Methods
				3.1. Isolation and Culture of Primary Osteoblasts from Neonatal Rat Calvaria
				3.2. Isolation and Culture of Primary Osteoblasts from the Long Bones of Neonatal Rats
				3.3. Bone Formation Assay
				3.4. Fixation and Staining
				3.5. Quantification of Mineralised Bone Formation
				3.6. Statistics
			4. Notes
			References
	Ch04_Isolation of Primary Avian Osteocytes
		Chapter 4: Isolation of Primary Avian Osteocytes
			1. Introduction
			2. Materials
				2.1. Fertilized Chicken Eggs
				2.2. Media and Solutions
				2.3. Monoclonal Antibody OB7.3
			3. Methods
				3.1. Tissue Dissection
				3.2. Isolation of OBmix
				3.3. Isolation of Osteocytes
			4. Notes
			References
	Ch05_Isolation of Mouse Osteocytes Using Cell Fractionation for Gene Expression Analysis
		Chapter 5: Isolation of Mouse Osteocytes Using Cell Fractionation for Gene Expression Analysis
			1. Introduction
			2. Materials
				2.1. Bone Fractionation and Sorting of GFP Positive Osteocytes by FACS
			3. Methods
				3.1. Bone Fractionation and Sorting of GFP Positive Osteocytes by FACS
			4. Notes
			References
	Ch06_Studying Osteocyte Function Using the Cell Lines MLO-Y4 and MLO-A5
		Chapter 6: Studying Osteocyte Function Using the Cell Lines MLO-Y4 and MLO-A5
			1. Introduction
			2. Materials
				2.1. Tissue Culture Reagents
				2.2. Fluid Flow Reagents
				2.3. Dendrite Quantitation Reagents
				2.4. siRNA Reagents
				2.5. E11 Western Blotting
			3. Methods
				3.1. General Maintenance of MLOY4 Osteocyte-Like Cell-Line
				3.2. Collagen Coating of Tissue Culture Treated Dishes
				3.3. Fluid Flow using Flexcell® Streamer® Shear Stress Device (Flexcell)
				3.4. Staining and Quantitation of Dendrite Length
				3.5. E11 siRNA Transfection of MLO-Y4
				3.6. General Maintenance of MLO-A5 Late Osteoblast/Early Osteocyte-Like Cell-Line
				3.7. Mineralization of MLO-A5
			4. Notes
			References
	Ch07_Isolation, Differentiation, and Characterisation of Skeletal Stem Cells from Human BM
		Chapter 7: Isolation, Differentiation, and Characterisation of Skeletal Stem Cells from Human Bone Marrow In Vitro and In Vivo
			1. Introduction
			2. Materials
				2.1. Equipment for Tissue Culture and Stromal Cell Isolation
				2.2. Equipment for In Vivo Diffusion Chamber Experiments
				2.3. Stromal Cell Isolation and Culture
				2.4. Stromal Cell Differentiation
				2.5. Alkaline Phosphatase Staining
				2.6. Alkaline Phosphatase Activity
				2.7. DNA Quantification
				2.8. Von Kossa Staining
				2.9. Safranin O Staining
				2.10. Immunostaining for Type II Collagen and SOX-9
				2.11. Oil Red O Staining
				2.12. Tissue Implant Models
			3. Methods
				3.1. Isolation of Bone Marrow Mononuclear Cells
				3.2. Antibody Labelling of STRO-1 Positive Cells
				3.3. Magnetic Separation of STRO-1 Positive Cells
				3.4. Osteogenic Differentiation of STRO-1 Positive Cells
				3.5. Chondrogenic Differentiation of STRO-1 Positive Cells
				3.6. Adipogenic Differentiation of STRO-1 Positive Cells
				3.7. Staining for Alkaline Phosphatase activity
				3.8. Analysis of Alkaline Phosphatase activity in cell lysates
				3.9. Measurement of Alkaline Phosphatase Activity
				3.10. Measuring DNA Concentration in Cell Lysates
				3.11. Von Kossa Staining for Bone Nodules and Osteoid
				3.12. Safranin O Staining of Chondrocytes Cultures
				3.13. Immunoloca- lisation of SOX-9 and Type II Collagen in Chondrocyte Cultures
				3.14. Oil Red O Staining
				3.15. Subcutaneous Implant Assay and Diffusion Chamber Assay
			4. Notes
			References
Part 2  Culture of osteoclasts
	Ch08_Rodent Osteoclast Cultures
		Chapter 8: Rodent Osteoclast Cultures
			1. Introduction
			2. Materials
			3. Methods
				3.1. Mouse Osteoclast Formation Assay
				3.2. Isolation of Mature Osteoclasts from Neonatal Rat Long Bones
				3.3. Fixation and Staining
				3.4. Quantification of Resorption
				3.5. Statistics
			4. Notes
			References
	Ch09_Isolation and Culture of Primary Chicken Osteoclasts
		Chapter 9: Isolation and Culture of Primary Chicken Osteoclasts
			1. Introduction
			2. Materials
				2.1. Tissue Culture Medium, Solutions, and Supplies
				2.2. Preparation of Antibody-Conjugated Magnetic Beads
				2.3. Fixation and TRAP Staining
				2.4. Fixation and Immunostaining
				2.5. Preparation of Devitalized Bone or Ivory Discs for Bone Pit Resorption Studies
				2.6. Preparation of Gold-Coated Glass Coverslips for Phagokinetic Motility Studies ( see Note 4)
			3. Methods
				3.1. Isolation of Osteoclasts from Calcium-Deficient Chicks ( see Note 5)
				3.2. Osteoclast Culture
				3.3. Assay Techniques
			4. Notes
			References
	Ch10_Isolation and Purification of Rabbit Osteoclasts
		Chapter 10: Isolation and Purification of Rabbit Osteoclasts
			1. Introduction
			2. Materials
				2.1. General Reagents
				2.2. Isolation and Purification of Rabbit Osteoclasts
				2.3. Generation of Rabbit Osteoclast-Like Cells
				2.4. Isolation of Rabbit Osteoclasts Using Immunomagnetic Beads
				2.5. Staining for Tartrate Resistant Acid Phosphatase
				2.6. Immunostaining for av b3 Integrin (Vitronectin Receptor)
				2.7. Detection of F-actin Rings
				2.8. Resorption Pit Assay
			3. Methods
				3.1. Isolation and Culture of Rabbit Osteoclasts
				3.2. Enrichment of Osteoclasts Using Foetal Calf Serum Gradients
				3.3. Purification of Rabbit Osteoclasts on Plastic (from Subheading  3.1)
				3.4. Generating Large Numbers of Rabbit Osteoclast-Like Cells in Vitro
				3.5. Isolation of Rabbit Osteoclasts Using Immunomagnetic Beads
				3.6. Characterisation of Rabbit Osteoclasts
			4. Notes
			References
	Ch11_Generation of Human Osteoclasts from Peripheral Blood
		Chapter 11: Generation of Human Osteoclasts from Peripheral Blood
			1. Introduction
			2. Materials
				2.1. Tissue Culture Reagents
				2.2. TRAcP Activity Reagents
				2.3. Staining
			3. Methods
				3.1. Isolation of PBMCs from Peripheral Blood or Buffy Coats
				3.2. Purification of CD14 + Osteoclast Precursor Cells
				3.3. Expansion of Macrophages/Osteoclast Precursors
				3.4. Cryopreservation of Osteoclast Precursors
				3.5. Generation of Osteoclasts for Assessment of Osteoclastogenesis
				3.6. Generation of Osteoclasts for Functional Studies
				3.7. Analysis of Osteoclast Formation
				3.8. Analysis of Osteoclast Activity
			4. Notes
			References
	Ch12_Osteoclast Formation in Mouse Co-cultures
		Chapter 12: Osteoclast Formation in Mouse Co-cultures
			1. Introduction
			2. Materials
				2.1. General Reagents/Materials
				2.2. Tissue Culture Reagents
				2.3. TRAcP Stain Reagents
			3. Methods
				3.1. Osteoblasts
				3.2. Isolation of Bone Marrow Cells
				3.3. Setting Up the Co-culture
				3.4. Collagen Gel Culture
				3.5. Purification of Osteoclasts Obtained by Co-culture
				3.6. Tartrate Resistant Acid Phosphatase Staining
				3.7. Quantification of the Resorption Area
			4. Notes
			References
	Ch13_RANKL-Mediated Osteoclast Formation from Murine RAW 264.7 cells
		Chapter 13: RANKL-Mediated Osteoclast Formation from Murine RAW 264.7 cells
			1. Introduction
			2. Materials
				2.1. Tissue Culture Medium, Solutions, and Supplies
			3. Methods
				3.1. Preparation of Devitalized Bone or Dentine Slices
				3.2. RAW 264.7 Cell Culture
				3.3. RAW-OC Formation ( See Note 4)
				3.4. Serum Gradient Purification of RAW-OC
				3.5. Phenotypic and Functional Characterization of RAW-OCs
			4. Notes
			References
Part 3  Biochemical and molecular analysis of bone cells
	Ch14_Transfection of Osteoclasts and Osteoclast Precursors
		Chapter 14: Transfection of Osteoclasts and Osteoclast Precursors
			1. Introduction
				1.1. Amaxa™ Nucleofector
				1.2. Lentiviral Vectors
				1.3. Adenoviral Vectors
			2. Materials
				2.1. Primary Cells
				2.2. Cell Lines for Virus Production
				2.3. Reagents
				2.4. Culture Media and Buffers
				2.5. Equipment
			3. Methods
				3.1. Transfecting Human Osteoclast Precursors Using the Amaxa™ Nucleofector
				3.2. Generating Lentivirus
				3.3. Transduction of Human Osteoclast Precursors with Lentiviral Vectors
				3.4. Generating Recombinant Adenoviruses
				3.5. Determining the Titre of Adenovirus
				3.6. Amplification of Adenoviral Stocks ( See Note 16)
				3.7. Adenovirus Transduction of Mature Human Osteoclasts
				3.8. Adenovirus Transduction of Mature Mouse Osteoclasts
				3.9. Assessment of Cell Viability
				3.10. Assessment of Transfection Efficiency
			4. Notes
			References
	Ch15_Analysis of Signalling Pathways by Western Blotting and Immunoprecipitation
		Chapter 15: Analysis of Signalling Pathways by Western Blotting and Immunoprecipitation
			1. Introduction
			2. Materials
				2.1. Immunopreci- pitation
				2.2. Western Blotting
			3. Methods
				3.1. Immunopreci- pitation
				3.2. Western Blotting
			4. Notes
			References
	Ch16_Analysis of Transcriptional Regulation in Bone Cells
		Chapter 16: Analysis of Transcriptional Regulation in Bone Cells
			1. Introduction
			2. Materials
				2.1. Promoter-Reporter Assay
				2.2. Nonradioactive Electrophoretic Mobility Shift Assay
				2.3. Chromatin Immunoprecipitation Assay
			3. Methods
				3.1. Dual Luciferase Reporter Assay
				3.2. Electrophoretic Mobility Shift Assay
				3.3. Chromatin Immunoprecipitation Assay
			4. Notes
			References
	Ch17_Extraction of Nucleic Acids from Bone
		Chapter 17: Extraction of Nucleic Acids from Bone
			1. Introduction
			2. Materials
				2.1. Equipment
				2.2. For DNA Extraction
				2.3. For RNA Extraction
				2.4. General Reagents
			3. Methods
				3.1. DNA Extraction from Fresh Bone
				3.2. DNA Extraction from Cultured Cells
				3.3. DNA Extraction from Dried or Embedded Bone
				3.4. RNA Extraction from Fresh Bone
				3.5. Extraction of RNA from Frozen Bone
				3.6. Extraction of RNA from Adherent Cultured Cells
				3.7. Extraction of RNA from Nonadherent Cultured Cells
				3.8. Quantification of Nucleic Acids by Spectrophotometry
				3.9. Evaluation of RNA Integrity by Agarose Gel Electrophoresis
			4. Notes
			References
	Ch18_Analysis of Gene Expression in Bone by Quantitative RT_PCR
		Chapter 18: Analysis of Gene Expression in Bone by Quantitative RT/PCR
			1. Introduction
				1.1. Assay Choice for Quantitative PCR
				1.2. Methods for Quantification of Transcripts
				1.3. Relating Expression of Target Genes to an Internal Control
				1.4. Preparation of Input RNA
			2. Materials
				2.1. Reverse Transcription
			3. Methods
				3.1. Reverse Transcription
				3.2. Designing Primers and Probes for Hydrolysis qPCR Assays
				3.3. Design of Primers for SYBR Green qPCR Assays
				3.4. Preparation of an Exogenous Control
				3.5. Preparation of an External Reference Curve for Absolute Quantification
				3.6. Preparation of cDNA Reference Curve for Relative Quantification
				3.7. Performing Quantitative RT-PCR Using Hydrolysis Probes
				3.8. Performing Quantitative RT-PCR Using SYBR Green Assays
				3.9. Analysis of q-RT-PCR Data
			4. Notes
			References
Part 4 Microscopical techniques
	Ch19_Histomorphometry in Rodents
		Chapter 19: Histomorphometry in Rodents
			1. Introduction
			2. Materials
				2.1. Sectioning, Fixation and Embedding
				2.2. Von Kossa and McNeal Stain
				2.3. Toluidine Blue Stain
				2.4. Tartrate Resistant Acid Phosphatase Stain
				2.5. Cement Line Stain
				2.6. Histomor-phometric Analysis
			3. Methods
				3.1. Fixation, Dehydration and Infiltration of Bone Samples
				3.2. Preparation of Glass Vials for Tissue Embedding
				3.3. Embedding for Routine Histology
				3.4. Embedding for Histochemistry and Immunohisto
chemistry
				3.5. Preparation of Micromilled Cross-Sections for Routine Histological Analysis
				3.6. Preparation of Micromilled Cross-Sections for Analysis of Fluorochrome Labels
				3.7. Von Kossa Stain
				3.8. Von Kossa/McNeal Stain
				3.9. Toluidine Blue Stain
				3.10. Tartrate Resistant Acid Phosphatase Staining
				3.11. Cement Line Stain
				3.12. Cancellous Bone Histomorphometry
				3.13. Assessment of Bone Structural Parameters
				3.14. Analysis of Dynamic Histomorphometry in Mice
				3.15. Assessment of Osteoclast Numbers and Bone Resorption in Mice
				3.16. Assessment of Bone Mineralization in Mice
				3.17. Assessment of Dynamic Histomorphometry in Rats
				3.18. Assessment of Static Histomorphometry in Rats
				3.19. Assessment of Remodeling-Based Parameters in Rats
				3.20. Cortical Bone Histomorphometry
				3.21. Periosteal and Endocortical Bone Formation
			4. Notes
			References
	Ch20_Studying Gene Expression in Bone by In Situ Hybridization
		Chapter 20: Studying Gene Expression in Bone by In Situ Hybridization
			1. Introduction
			2. Materials
				2.1. Tissue Collection, Fixation, Decalcification, and Embedding
				2.2. Tissue Sectioning
				2.3. Generation of DIG-Labeled Riboprobes
				2.4. Hybridization, Washes, Probe Detection, and Signal Development
			3. Methods
				3.1. Tissue Collection, Fixation, Decalcification, and Embedding
				3.2. Cryo-Sectioning
				3.3. Linearization of the Plasmid Containing the Probe of Interest
				3.4. Generation of DIG-Labeled Riboprobes
				3.5. Hybridization
				3.6. Washes, Probe Detection, and Signal Development
			4. Notes
			References
	Ch21_Immunostaining of Skeletal Tissues
		Chapter 21: Immunostaining of Skeletal Tissues
			1. Introduction
			2. Materials
			3. Methods
				3.1. Preparation of Mouse Knee Joint Paraffin Blocks
				3.2. Immuno-histochemical Staining Using Enzyme-Conjugated Antibodies
				3.3. Immuno-fluorescence Staining Using Fluorochrome-Conjugated Antibodies
			4. Notes
			References
	Ch22_Techniques for the Study of Apoptosis in Bone
		Chapter 22: Techniques for the Study of Apoptosis in Bone
			1. Introduction
				1.1. Toluidine Blue Staining
				1.2. LDH Assay
				1.3. Nick Translation
				1.4. DNA Laddering
				1.5. Caspase Activation
			2. Materials
				2.1. General Materials
				2.2. Toluidine Blue Staining
				2.3. LDH Assay
				2.4. Nick Translation
				2.5. DNA Laddering
				2.6. Caspase 3-7 Detection
			3. Methods
				3.1. Detection of Apoptosis by Toluidine Blue Staining
				3.2. Assessment of Cell Viability Using the LDH Assay
				3.3. Detection of Fragmented DNA Using Nick Translation
				3.4. Detecting of DNA Laddering in Cells and Tissue Sections
				3.5. Detection of Caspase Activation
			4. Notes
			References
	Ch23_Transmission Electron Microscopy of Bone
		Chapter 23: Transmission Electron Microscopy of Bone
			1. Introduction
			2. Materials
				2.1. Fixative
				2.2. Osmium and Ferrocyanide Postfixative
				2.3. Osmium and Cacodylate Postfixative
				2.4. Decalcification Solution
				2.5. Goldner’s Masson Trichrome
				2.6. Methylene Blue
				2.7. Von Kossa
				2.8. Uranyl Acetate
				2.9. Lead Nitrate
				2.10. Epoxy Resin
			3. Methods
				3.1. Perfusion Fixation of Animal Bones
				3.2. Immersion Fixation of Animal Bones
				3.3. Immersion Fixation of Human Bone Samples
				3.4. Immersion Fixation of Cultured Mineralized Tissues
				3.5. Decalcification of Mineralized Tissues
				3.6. General Approach to Embedding and Analysis of Mineralized Tissues by TEM
				3.7. Embedding of Small Tissue Samples
				3.8. Embedding of Larger Bone Samples
				3.9. Sectioning Mineralized Tissues
				3.10. Methylene Blue Staining of Semi-Thin Sections
				3.11. Modified Goldner’s Masson Stain
				3.12. Von Kossa
				3.13. Staining of Ultrathin Sections with Uranyl Acetate
				3.14. Staining of Ultrathin Sections with Lead Nitrate ( 3)
			4. Notes
			References
	Ch24_Scanning Electron Microscopy of Bone
		Chapter 24: Scanning Electron Microscopy of Bone
			1. Introduction
				1.1. Instrumentation and Imaging Modes
				1.2. Types of Samples and Interpretation of SEM Data
			2. Materials
				2.1. Chemicals
				2.2. Instrumentation
				2.3. Software
			3. Methods
				3.1. Cutting Bone and Bones
				3.2. Fixation of Cells or Tissue for SEM
				3.3. Drying Samples
				3.4. Coating
				3.5. Morphological Imaging of Cells on Bone in 3D
				3.6. Examining Undersides of Bone Cells in Contact with Substrate
				3.7. Morphological Imaging of Bone Alone in 3D
				3.8. Bone and Dentine Slices Etched by Osteoclasts In Vitro
				3.9. BSE SEM Imaging Methods
				3.10. BSE Imaging of Embedded Bone
				3.11. Correlation of SEM with Other Imaging Means
				3.12. Shipping Bone Samples
			4. Notes
			References
	Ch25_Fluorescence Imaging of Osteoclasts Using Confocal Microscopy
		Chapter 25: Fluorescence Imaging of Osteoclasts Using Confocal Microscopy
			1. Introduction
				1.1. Principles of Confocal Microscopy
				1.2. The Use of Confocal Microscopy in Osteoclasts
			2. Materials
				2.1. Equipment
				2.2. General Reagents
				2.3. Reagents for Synthesising Fluorescent BPs
				2.4. Useful Probes for Staining Osteoclast Structures
				2.5. Software for Image Analysis
			3. Methods
				3.1. Synthesis of Fluorescently Labelled Alendronate
				3.2. Culture of Osteoclasts for Confocal Microscopy
				3.3. Staining of Osteoclasts
				3.4. Confocal Microscopy of Osteoclasts
				3.5. Visualisation of the Data Post-acquisition
				3.6. Generating Three-Dimensional Data Sets
				3.7. Visualising Three-Dimensional Data Sets
				3.8. Spectral Imaging
				3.9. Live Cell Imaging Using Confocal Microscopy
				3.10. Advanced Uses and Probes for Confocal Microscopy
				3.11. Non-confocal Methods for Achieving Optical Sectioning of a Specimen
			4. Notes
			References
	Ch26_Live Imaging of Bone Cell and Organ Cultures
		Chapter 26: Live Imaging of Bone Cell and Organ Cultures
			1. Introduction
				1.1. General Considerations for Live Imaging of Cultured Bone Cells and Bone Organ Cultures
				1.2. General Considerations About Equipment
				1.3. Image Quality Versus Phototoxicity
				1.4. Selection of Imaging Fields
				1.5. Selection of Time Intervals
				1.6. Types of Probes and Their Limitations
				1.7. Focal Drift
				1.8. Data Storage and Data Handling
			2. Materials
				2.1. Animals, Cells, and Tissues
				2.2. Cell and Tissue Culture Reagents
				2.3. Other Buffers, Solutions, and Reagents
				2.4. Equipment
			3. Methods
				3.1. Preparation and Culture of Primary Osteoblasts
				3.2. Preparation of Neonatal Mouse Calvaria
				3.3. Time-Lapse Imaging Techniques
				3.4. Post-acquisition Processing of Image Stacks, Generation of Movies and Quantitation of Dynamic Events
			4. Notes
			References
Part 5 Imaging techniques
	Ch27_Analysis of Bone Architecture in Rodents Using Microcomputed Tomography
		Chapter 27: Analysis of Bone Architecture in Rodents Using Microcomputed Tomography
			1. Introduction
				1.1. Micro-CT Analysis
				1.2. Acquiring the X-Ray Projection Images
				1.3. Image Reconstruction
				1.4. Analysis of the 3D Image Stack
			2. Materials
				2.1. m CT Scanner
				2.2. Computing Equipment
				2.3. Other Materials
			3. Methods
				3.1. Ex Vivo Analysis of Bone Architecture
				3.2. Analysis of Focal Bone Lesions Using m CT
				3.3. In Vivo m CT Analysis
			4. Notes
			References
	Ch28_Bone Measurements by Peripheral Quantitative Computed Tomography in Rodents
		Chapter 28: Bone Measurements by Peripheral Quantitative Computed Tomography in Rodents
			1. Introduction
			2. Materials
			3. Methods
				3.1. Experimental Design and Choice of Animals
				3.2. Skeletal Regions of Interest
				3.3. Quality Assurance
				3.4. Limb Positioning
				3.5. Instrument Setting
				3.6. Data Analysis and Interpretation
				3.7. Interpretation of Data
				3.8. Analysis of Scan Data
			4. Notes
			References
	Ch29_Quantitative X-ray Imaging of Rodent Bone by Faxitron
		Chapter 29: Quantitative X-ray Imaging of Rodent Bone by Faxitron
			1. Introduction
			2. Materials
				2.1. General Reagents/Materials
				2.2. Equipment and Computer Software
			3. Methods
				3.1. Dissection and Preparation of Skeletal Elements
				3.2. Faxitron Digital X-ray Imaging of Excised Bones
				3.3. Image Processing
				3.4. Generating Pseudo-coloured Images of Individual Bones
				3.5. Generating a Montage of Pseudo-coloured Images of All Bones
				3.6. Processing and Cleaning Montages for Analysis of Mineralisation
				3.7. Determination of Relative Mineral Content
			4. Notes
			Reference
	Ch30_Bioluminescence Imaging of Bone Metastasis in Rodents
		Chapter 30: Bioluminescence Imaging of Bone Metastasis in Rodents
			1. Introduction
				1.1. Optical Imaging
				1.2. Fluorescence Imaging
				1.3. Bioluminescence Imaging
				1.4. Models of Skeletal Metastases
			2. Materials
			3. Methods
				3.1. Induction of Bone Metastases by Intra-cardiac Injection of Cancer Cells
				3.2. Induction of Bone Metastases by Direct Intra-Osseous Inocculation of Cancer Cells
				3.3. Bioluminescence Imaging Using the IVIS Spectrum System
				3.4. Quantification of the Bioluminescent Signal
			4. Notes
			References
	Ch31_Fourier Transform Infrared Imaging of Bone
		Chapter 31: Fourier Transform Infrared Imaging of Bone
			1. Introduction
			2. Materials
				2.1. Equipment and Software
				2.2. Tissue Sections
			3. Methods
				3.1. Preparation of Section
				3.2. Decide upon Which Type of Analysis is Required
				3.3. Select the Area to be Analyzed
				3.4. Acquisition of Spectral Images
				3.5. Analysis of Individual Spectra
				3.6. Analysis of Whole Spectral Images
			4. Notes
			References
	Ch32_Raman Microscopy of Bone
		Chapter 32: Raman Microscopy of Bone
			1. Introduction
			2. Materials
			3. Methods
				3.1. Instrument Calibration
				3.2. Sample
				3.3. Acquiring Spectra
				3.4. Pre-processing
				3.5. Analysis
				3.6. Principal Component Analysis
				3.7. Polarisation/Orientation
			References
Part 6 In vivo techniques
	Ch33_The Calvarial Injection Assay
		Chapter 33: The Calvarial Injection Assay
			1. Introduction
			2. Materials
				2.1. Injection
				2.2. Tissue Processing
				2.3. TRAcP/Von Kossa/Light Green Stain
				2.4. Goldner’s Trichrome Stain
			3. Methods
				3.1. Injection Protocol (Resorption)
				3.2. Tissue Processing
				3.3. TRAcP/Von Kossa/Light Green Staining of Mouse Calvariae
				3.4. Goldner’s Trichrome
				3.5. Analysis of Results
			4. Notes
			References
	Ch34_Ovariectomy/Orchidectomy in Rodents
		Chapter 34: Ovariectomy/Orchidectomy in Rodents
			1. Introduction
			2. Materials
				2.1. Surgical Instruments
				2.2. Anaesthetic Agents
			3. Methods
				3.1. Animal Husbandry
				3.2. Anaesthesia
				3.3. Preoperative Care
				3.4. Operative Technique for Ovariectomy
				3.5. Operative Technique for Orchidectomy
				3.6. Post-operative Care
			4. Notes
			References
Part 7 Mechanical loading techniques
	Ch35_Mechanical Properties of Bone Ex Vivo
		Chapter 35: Mechanical Properties of Bone Ex Vivo
			1. Introduction
			2. Materials
			3. Methods
				3.1. General Considerations for the Preparation of Bone Samples
				3.2. Preparing Prisms of Cortical Bone
				3.3. Preparing Cancellous Bone Cores
				3.4. Preparation of Mouse Bones
				3.5. General Considerations for Mechanical Testing
				3.6. Repeating Tests on the Same Specimens
				3.7. Types of Mechanical Testing
				3.8. Tension Testing
				3.9. Analysing Data from Tension Testing
				3.10. Compression Testing
				3.11. Bending
				3.12. The Elastic Modulus of Cortical Bone
				3.13. Density of Bone by Archimedes’ Principle
				3.14. Speed of Sound Measured Using Ultrasound
				3.15. Ashing
			4. Notes
			References
	Ch36_Mechanical Stimulation of Bone Cells Using Fluid Flow
		Chapter 36: Mechanical Stimulation of Bone Cells Using Fluid Flow
			1. Introduction
			2. Materials
				2.1. Tissue Culture Media and Solutions
				2.2. Instruments
				2.3. Pumps
				2.4. Flow Chamber
				2.5. Incubator
				2.6. Microscope
			3. Methods
				3.1. Preparation of Cells
				3.2. Seeding of Cells
				3.3. Assembly of the Flow Apparatus
				3.4. Analysis of the Cellular Response to Fluid Flow
			4. Notes
			References
	Ch37_Using Cell and Organ Culture Models to Analyze Responses to Mechanical Stimulation
		Chapter 37: Using Cell and Organ Culture Models to Analyze Responses of Bone Cells to Mechanical Stimulation
			1. Introduction
				1.1. Limitations of In Vitro Organ and Cell Culture Strain Application Models
				1.2. The Effects of Loading: Mechanical Strain, Fluid Shear, and Streaming Potentials
				1.3. In Vitro Cell Culture Loading Models
				1.4. Biaxial Straining
				1.5. Uniaxial Straining
			2. Materials
				2.1. Tissue Culture Media
			3. Methods
				3.1. An Example of Four-Point Bending of Monolayers of Cells on Plastic Strips
				3.2. In Vitro Organ Culture Loading Models
				3.3. Rat Ulnae Organ Cultures ( see Notes 5 and 6)
				3.4. Loadable Rat Calvaria Organ Cultures ( see Notes 5 and 6)
				3.5. Loading of the Chick Tibiotarsus ( see Notes 5, 6 , and 8)
				3.6. Longer-Term Perfusion Loading Models
			4. Notes
			References
	Ch38_In Vivo Mechanical Loading
		Chapter 38: In Vivo Mechanical Loading
			1. Introduction
				1.1. Mechano-Adaptation
				1.2. Loading Models
				1.3. A ssessment of Adaptive Modelling and Remodelling In Vivo
				1.4. Determination of the In Vivo Mechanical Environment of Bone
			2. Materials
				2.1. Measurement of Bone Strains In Vivo
			3. Methods
				3.1. Strain Gauge Preparation
				3.2. Prepare the Bones Ex Vivo
				3.3. Prepare the Mouse In Vivo
				3.4. Attach the Gauge
				3.5. Measure Strains
				3.6. Application of Bone Loads In Vivo
				3.7. Mechanical Loading of the Tibia/Ulna
			4. Notes
			References
xx_back-matter
	INDEX
                        
Document Text Contents
Page 2

M E T H O D S I N M O L E C U L A R B I O L O G Y ™

Series Editor
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University of Hertfordshire

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Page 328

314 I. Kramer et al.

9. Wash the slides in PBS for 3 min.
10. Remove the slides from the rack, and lay horizontally in a tray

humidifi ed with water-soaked paper towels. Add 800 μ l hybrid-
ization solution to each slide and incubate for between 1 and
3 h.

11. Add suffi cient DIG-labeled riboprobe to 1 ml of hybridization
solution to give a fi nal RNA concentration of 1.0 μ g/ml.

12. Heat the diluted riboprobe/hybridization solution to 80°C
for 5 min and transfer immediately to ice for 1 min. Remove
from the ice and maintain at room temperature until adding to
the slides.

13. Place a folded paper towel soaked in 50% formamide solution
at the bottom of a small plastic slide box to serve as a humidi-
fi ed hybridization cassette (see Note 21).

14. Remove the prehybridization solution and add 250 μ l of the
riboprobe/hybridization solution over the entire surface of the
slide. Carefully place a glass coverslip on top of the slide cover-
ing all tissue sections, avoiding entrapment of air bubbles.

15. Place the slides horizontally in the prepared humidifi ed plastic
box. Tightly seal the cassette with tape to avoid evaporation
overnight, while keeping the box upright to prevent leakage of
the hybridization solution. Place the box standing, i.e., with
the slides orientated horizontally and parallel to the ground in
a hybridization oven preset to an appropriate temperature (see
Note 22) and incubate overnight.

1. Heat 5× SSC and 0.2× SSC solutions to 70°C in covered-glass
staining dishes with empty slide racks in a water bath (see
Note 23).

2. Transfer the slides with coverslips to the 5× SSC solution and
incubate for 5 min at 70°C.

3. Transfer the slides one by one from the 5× SSC to the 0.2×
SSC solution. (The coverslips should automatically come off at
this point and remain in the 5× SSC solution).

4. Incubate slides at 70°C in 0.2× SSC for 1 h.
5. Remove the entire 0.2× SSC staining dish with the slides from

the water bath and allow to cool for 5 min at room
temperature.

6. Transfer the slides to B1 buffer and incubate for 5 min at room
temperature.

7. Lay the slides horizontally in a humidifi ed chamber, cover with
800 μ l 2% blocking solution, and incubate for 1 h at room
temperature.

3.6. Washes, Probe
Detection, and Signal
Development

Page 329

31520 Studying Gene Expression in Bone by In Situ Hybridization

8. Aspirate the blocking solution from the slides, replace with
700 μ l of diluted anti-DIG-AP antibody solution, and incu-
bate for 3 h at room temperature or overnight at 4°C in a
humidifi ed tray.

9. Aspirate the antibody solution from each slide and transfer all
slides to a slide rack.

10. Wash three times for 5 min in B1 buffer in a staining container
at room temperature.

11. Transfer to B3 buffer and incubate for 5 min at room
temperature.

12. Lay slides horizontally in a humidifi ed chamber and add 0.9 ml
0.1% Tween-20/B3 solution each slide.

13. Aspirate the Tween-20/B3 buffer and replace with 600 μ l B4
developing solution.

14. Cover the humidifi ed chamber with aluminum foil to block
out the light and incubate at room temperature for up to 12 h,
monitoring progression of signal development periodically
under a dissecting microscopy (see Note 24).

15. When the signal has developed to the desired level, stop the
reaction by transferring the slide to a slide rack in a staining
container fi lled with TE buffer. Incubate at room temperature
for 5 min.

16. Wash the slides in distilled water for 10 min at room tempera-
ture and air dry for 10–20 min.

17. Mount slides by adding about 350 μ l Kaiser’s glycerin gelatin
in a drop-by-drop fashion to each slide and store at 4°C.



1. PFA is toxic and should always be handled under a fume hood
while wearing personal protective equipment.

2. Dissolve the EDTA by adjusting the pH to 7.4 using 14.8 M
ammonium hydroxide rather than sodium hydroxide solution,
as EDTA dissolved in ammonium hydroxide solution has been
shown to result in more effi cient decalcifi cation of bone sam-
ples ( 6 ) .

3. Plasmid with cloned cDNA or part of a genomic coding
sequence. Suitable plasmids with recognition sites for T7, T3,
or SP6 polymerase for generating riboprobes are commercially
available via different sources. Expressed sequence tags (ESTs,
complete cDNAs and cDNA fragments) can be obtained from
the I.M.A.G.E. (Integrated Molecular Analysis of Genomes

4. Notes

Page 656

642

BONE RESEARCH PROTOCOLS

Index

Renilla luciferase ...................................................... 235–237
Resorption,

measurement of calcium ............................................171
measurement of collagen breakdown, CTX-I ............171

Resorption pit assay
analysis by confocal microscopy .................................418
analysis by reticle .......................................................138
analysis by SEM ................................................ 381–383
biochemical assays .....................................................171
counting stained pits with reflected

light ............................................... 108–109, 115
counting stained pits with transmitted

light ....................................................... 155, 171
importance of pH .............................................. 109–112
measuring resorbed area

by dot counting system ........................................115
by automated image analysis ........................ 182–183

method ............................... 107–109, 138–139, 155, 171
Reverse transcription (RT) ..............................................265
Reynolds number .............................................................590
RNA

extraction ..................................................... 62, 249–259
from cultured cells ....................................... 254–255
from fresh bone ............................................ 253–254
from frozen bone .................................................254

quantification ..................................................... 255–256

S

Safranin O stain .............................................. 86, 89, 93–94
SaOS-2 cell line ...................................................................6
Scanning electron microscopy (SEM) ..................... 365–399

backscattered electron imaging of resin embedded
bone ....................................................... 387–389

coating of samples ......................................................376
correlation with other imaging methods ............ 391–394
drying of samples ............................................... 374–376
general principles ............................................... 365–367
making casts of resin embedded bone ................ 389–390
optimising image display ................................... 383–387
removal of organic material ............................... 377–381

SDS polyacrylamid gel ............................................ 228–230
Sectioning

cryostat .............................................................. 310, 336
epoxy embedded bone................................................357
MMA embedded bone ..............................................280

Serum gradient
for purification of osteoclasts .....................152, 192–195,

199, 200
Shear stress. See also Fluid flow

calculation ..................................................................590
Shipping bone samples ....................................................394
SOX-9 staining ........................................................... 94–95
Spectral imaging ...................................................... 418–419
Speed of sound ................................................ 558, 567–568

Stains/staining methods
Alcian blue ............................................................. 86, 97
Celestine Blue ............................................................543
DAPI ........................................................ 123, 150, 154
Diff-Quick ................................................................122
Fast green ....................................................................94
Goldner’s Masson trichrome .....................................353
Haematoxylin QS ......................................................327
Lead nitrate for TEM ........................................ 354–360
Light Green ................................................... 86, 95, 541
Mayer’s haematoxylin ................................................282
Methylene blue .................................................. 353, 359
Oil Red O .............................................................. 87, 95
Safranin O ............................................................. 89, 93
Sirius red ......................................................................97
Sytox Green ....................................................... 154, 408
Toluidine blue ............................................ 108, 282, 287
TO-PRO-3 ....................................................... 209, 408
Uranyl acetate for TEM .................................... 354, 359
Von Kossa ............................. 85, 287, 354, 359, 540–541
Weigert’s haematoxylin .................................. 86, 93, 538

Stiffness . See also Mechanical testing ...............................566
Strain

biaxial ................................................................ 599–601
definition ...................................................................621
parameters .................................................................622
magnitude ..................................................................597
measurements ............................................................625
uniaxial .............................................................. 601–602

Strain gauges ....................................612–613, 625, 627–630
Streaming potentials. See Fluid flow
STRO-1. See Antibodies
Structural parameters of bone ..........................................290

T

2T3 cell line .....................................................................433
Tartrate-resistant acid phosphatase (TRAcP)

concentration of tartrate ............................................185
fast-garnet method ....................................................122
fast red method .................................................. 287–288
kit .............................................................. 106, 181–182
pararosanilin method ........................................ 149–150,

154–155, 179
quantitative colorimetric assay ........................... 163, 170
staining of GMA-embedded tissue ................... 540–541

TE85 cell line .............................................................. 6, 236
Tension testing ........................................................ 563–564
Tergazyme treatment for SEM imaging ..........................378
Three point loading ................................................. 562–563
Tibia, in vivo loading model ............................................630
Toluidine blue stain ......................................... 106, 282, 287
Transfection ............................................................. 205–221

by Amaxa nucleofector ...................................... 211–212
by MATra magnetic system ............................... 234, 236

Page 657

BONE RESEARCH PROTOCOLS


643
Index

with adenovirus .................................................. 216–217
with lentivirus ............................................................213
with siRNA ...............................................................207

Transmission electron microscopy ........................... 351–363
choice of buffer ..........................................................361
decalcification ............................................................356
embedding .................................................................357
fixation ............................................................... 354–355
sectioning ...................................................................357
staining of semi-thin sections ............................ 357–359
staining of ultra-thin sections ............................ 359–360

U

Ultrasound ...................................................... 558, 567–568
Uniaxial straining .................................................... 603–605
Uranyl nitrate stain .................................................. 354, 359

V

Vit C. See L-ascorbic acid
Vitronectin receptor. See Antibodies
Von Kossa stain .......................... 85, 287, 354, 359, 540–541
Von Kossa/McNeal stain ......................................... 286–287

W

Weigert’s haematoxylin ........................................ 86, 93, 538
Weightlessness .................................................................603
Western blotting ...................................................... 228–230
Wheat germ agglutinin ...................................................408

X

XCT scanners. See pQCT
X-ray imaging .......................................................... 409–506

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