Analysis of Mouse Growth Plate Development

Laura Mangiavini1, Christophe Merceron2, Ernestina Schipani1

1 Department of Orthopaedic Surgery, Department of Medicine, Division of Endocrinology and Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, Michigan, 2 Faculty of Dental Surgery, LUNAM, Nantes University, Nantes
Publication Name:  Current Protocols in Mouse Biology
Unit Number:   
DOI:  10.1002/9780470942390.mo150094
Online Posting Date:  March, 2016
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Abstract

To investigate skeletal development, pathophysiological mechanisms of cartilage and bone disease, and eventually assess innovative treatments, the mouse is a very important resource. During embryonic development, mesenchymal condensations are formed, and cells within these mesenchymal condensations either directly differentiate into osteoblasts and give origin to intramembranous bone, or differentiate into chondrocytes and form a cartilaginous anlage. The cartilaginous anlage or fetal growth plate is then replaced with bone. This process is also called endochondral bone development, and it is responsible for the generation of most of our skeleton. Here we discuss in detail the most common in vivo and in vitro techniques our laboratory is currently using for the analysis of the mouse fetal growth plate during development. © 2016 by John Wiley & Sons, Inc.

Keywords: endochondral bone development; cartilage; mouse; cell culture; staining

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Mouse Embryo Harvesting for Paraffin Sections
  • Support Protocol 1: Orientation of Paraffin‐Embedded Specimens for Growth Plate Sectioning
  • Basic Protocol 2: Mouse Embryo Harvesting for Freshly Frozen Sections
  • Alternate Protocol 1: Mouse Embryos Harvesting for Fixed Frozen Sections
  • Basic Protocol 3: Alizarin Red/Alcian Blue Whole‐Mount Staining
  • Basic Protocol 4: Hematoxylin and Eosin Staining on the Fetal Growth Plate
  • Basic Protocol 5: Safranin O Staining to Evaluate GAGs in Mouse Growth Plate
  • Basic Protocol 6: In Situ Hybridization: A Method to Detect mRNA In Vivo
  • Basic Protocol 7: In Situ Cell Death Detection (TUNEL Assay)
  • Basic Protocol 8: Immunohistochemistry on Formalin‐Fixed Paraffin‐Embedded Tissues
  • Basic Protocol 9: Proliferation Assays: BrdU Assay to Evaluate Cell Proliferation
  • Support Protocol 2: BrdU Detection with a Fluorescent Dye
  • Alternate Protocol 2: EdU Assay to Evaluate Cell Proliferation
  • Basic Protocol 10: Proliferating Cell Nuclear Antigen (PCNA) Assay
  • Support Protocol 3: PCNA Detection with a Fluorescent Dye
  • Basic Protocol 11: EF5 Staining: A Method to Detect Hypoxia in the Developing Growth Plate
  • Basic Protocol 12: Quantification of Fluorescent Signal‐Labeled Cells Using Image J
  • Alternate Protocol 3: Quantification of Signal‐Labeled Cells on Brightfield Pictures Using Image J
  • Basic Protocol 13: In Vitro Recombination: A Tool to Study Growth Plate Development
  • Support Protocol 4: 10X Collagenase Type II Stock Solution Preparation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Mouse Embryo Harvesting for Paraffin Sections

  Materials
  • Pregnant female mouse
  • Isoflurane or CO 2 chamber
  • Ice
  • Phosphate‐buffered saline (PBS), 1×, sterile (see recipe)
  • 70% (v/v) ethanol (EtOH; see recipe)
  • 4% paraformaldehyde (PFA; see recipe)
  • 80% (v/v) EtOH (see recipe)
  • 95% (v/v) EtOH (see recipe)
  • 100% (v/v) EtOH
  • Xylene
  • Paraffin (Fisherbrand Paraplast X‐Tra Tissue Embedding Medium; Fisher Scientific, cat. no. 23‐021‐401)
  • Surgical instruments including:
    • Sterile scalpels
    • Scissors
    • Forceps
    • Microdissecting forceps and scissors
  • 100‐mm petri dishes
  • 25‐ml glass scintillation vials
  • Stereomicroscope
  • Microsette biopsy cassettes
  • Automated processor
  • Embedding station
  • Molds (plastic or metallic)
  • Microtome
  • Paraffin blocks
  • Disposable low profile blades
  • Flat ice block
  • Paintbrush
  • Water bath
  • Superfrost Plus slides
  • Paper towels
  • Slide boxes
  • Magnifying glass
  • Plastic or glass containers

Support Protocol 1: Orientation of Paraffin‐Embedded Specimens for Growth Plate Sectioning

  Additional Materials (also see protocol 1)
  • OCT Compound (Fisher Healthcare Tissue‐Plus; Fisher Scientific, cat. no. 4585)
  • Flat block of dry ice
  • Sample holder
  • Forceps
  • Cryostat
  • High profile blade
  • −80°C freezer
  • Paintbrush
  • Superfrost Plus slides
  • Slide box

Basic Protocol 2: Mouse Embryo Harvesting for Freshly Frozen Sections

  Materials
  • 30% sucrose/PBS (see recipe)
  • 1×phosphate‐buffered saline (PBS; see recipe)
  • Glass or plastic container
  • Rocking platform

Alternate Protocol 1: Mouse Embryos Harvesting for Fixed Frozen Sections

  Materials
  • Pregnant female mouse
  • 1× phosphate‐buffered saline (PBS; see recipe)
  • 95% EtOH (see recipe)
  • 100% acetone
  • Alizarin Red/Alcian Blue staining working solution (see recipe)
  • Distilled water
  • 1% KOH working solution (see recipe)
  • 20% glycerol/1% KOH (see recipe)
  • 50% glycerol/1% KOH (see recipe)
  • 80% glycerol/1% KOH (see recipe)
  • 100% glycerol
  • 100‐mm petri dishes
  • Scissors and forceps
  • 25‐ml glass scintillation vials
  • 37°C incubator

Basic Protocol 3: Alizarin Red/Alcian Blue Whole‐Mount Staining

  Materials
  • 5‐μm paraffin‐embedded tissue sections on Superfrost Plus slides (see protocol 1)
  • Xylene in Coplin jars
  • 100% EtOH in Coplin jars
  • 95% EtOH in Coplin jars (see recipe)
  • dH 2O in Coplin jars
  • Harris Hematoxylin in Coplin jars
  • Eosin Y in Coplin jars
  • Cold tap water
  • Xylene‐based mounting medium
  • Slide warmer
  • Glass coverslips
  • Slide racks
  • Slide tray

Basic Protocol 4: Hematoxylin and Eosin Staining on the Fetal Growth Plate

  Materials
  • 5‐μm paraffin‐embedded tissue sections on Superfrost Plus slides (see protocol 1)
  • Xylene in Coplin jars
  • 100% ethanol in Coplin jars
  • 95% ethanol in Coplin jars (see recipe)
  • dH 2O in Coplin jars
  • Weigert's Iron Hematoxylin working solution (see recipe)
  • 0.1% Fast Green solution (see recipe)
  • 1% acetic water (see recipe #18)
  • 0.08% Safranin O solution (see recipe)
  • Xylene‐based mounting medium
  • Slide warmer
  • Wheaton glass staining dishes or Coplin jars
  • Transfer pipets
  • Paper filters
  • Glass coverslips
  • Slide racks
  • Slide tray

Basic Protocol 5: Safranin O Staining to Evaluate GAGs in Mouse Growth Plate

  Materials
  • 5× transcription buffer (Promega, cat. no. P118B)
  • 100 mM dithiothreitol (DTT; Promega, cat. no. P117B)
  • rATP (Promega, cat. no. P113B)
  • rCTP (Promega, cat. no. P114B)
  • rGTP (Promega, cat. no.P115B)
  • 35 S‐UTP (Perkin Elmer, cat. no. NEG739H001MC)
  • RNase inhibitor (Promega, cat. no. N211A)
  • Linear DNA template of target gene
  • RNA polymerase (T3, T7 or Sp6; Fisher, cat. no. BP3206–1; Promega, cat. no. P207B‐ P108B)
  • Ice
  • DNase I (Promega, cat. no. M610A)
  • 0.5 M EDTA, pH 8.0
  • 5× NTE (see recipe)
  • DEPC‐treated water
  • Scintillation fluid (e.g, Scintiverse)
  • 4% paraformaldehyde (PFA; see recipe)
  • RNase away
  • Xylene
  • 100% EtOH
  • 95% EtOH (see recipe)
  • 1× phosphate‐buffered saline (PBS; see recipe)
  • 10 μg/ml proteinase K working solution (see recipe)
  • 0.2 N hydrochloric acid (HCl; see recipe)
  • dH 2O
  • 0.1 M triethanolamine/30 mM acetic anhydride (TEA/AA), pH 7.5 (see recipe)
  • 70% EtOH (see recipe)
  • UltraPure formamide (Sigma, cat. no. 47671)
  • Hybridization solution (see recipe)
  • 1 M dithiothreitol (DTT; see recipe)
  • 2× SSC (see recipe)
  • 1× saline‐sodium citrate (SSC; see recipe)
  • 4× SSC/formamide (see recipe)
  • 20 mg/ml RNase A (see recipe)
  • 70% EtOH/0.1× SSC (see recipe)
  • 80% EtOH/0.1× SSC (see recipe)
  • 95% EtOH/0.1× SSC (see recipe)
  • Kodak Autoradiography Emulsion NTB 2 (VWR Scientific, cat. no. IB8895666)
  • 2% glycerol working solution (see recipe)
  • Silica Gel Tel‐Tale (desiccant; Fisher Scientific, cat. no. S161)
  • Kodak developer solution (see recipe)
  • Kodak fixer solution (see recipe)
  • Harris Hematoxylin in Coplin jars
  • Tap water
  • dH 2O in Coplin jars
  • 95% EtOH in Coplin jars (see recipe)
  • Eosin Y in Coplin jars
  • 100% EtOH in Coplin jars
  • Xylene in Coplin jars
  • Windex
  • Geiger counter
  • Water baths at 37°, 52°, 85°, and 45°C
  • Vortex mixer
  • Probequant G‐50 columns (GE, cat. no. 28‐9034‐08)
  • Benchtop centrifuge
  • RNase‐free microcentrifuge tubes
  • 25‐ml glass scintillation vials
  • Liquid scintillation counter (LSC)
  • Slide warmer
  • 600‐ml Wheaton glass staining dishes (at least five to six)
  • Stainless steel slide racks
  • Humidified hybridization chamber
  • Absorbent underpads
  • Whatman paper
  • Parafilm
  • Hybridization oven at 55°C
  • Sterile‐filtered tips
  • 600‐ml Wheaton glass staining dishes with lids (at least 10; use different dishes from the prehybridization treatment)
  • Microwave
  • Autoradiography cassettes
  • Clear tape
  • Kodak Biomax MR single emulsion films (Kodak, cat. no. 870 1302)
  • Darkroom
  • Blade
  • Opaque plastic bottles
  • Heavy‐duty aluminum foil
  • 1‐liter glass beakers
  • Dip miser (Electron Microscopy Science, cat. no. 70520) – cleaned before use
  • Metal stand for the dip miser (Electron Microscopy Science)
  • 35‐mm petri dishes
  • Tape
  • Slides boxes (for 25 slides)
  • 50‐ml Falcon tubes
  • Paper towels
  • Slide draining rack
  • Glass coverslips
  • Kimwipes
  • Slide tray
  • Microscope with darkfield capacity
NOTE: Clean all work surfaces with RNase Away.

Basic Protocol 6: In Situ Hybridization: A Method to Detect mRNA In Vivo

  Materials
  • 5‐μm paraffin‐embedded tissue sections on Superfrost Plus slides (see protocol 1)
  • 1× phosphate‐buffered saline (PBS; see recipe)
  • Permeabilization solution (see recipe)
  • Xylene in Coplin jars
  • 100% EtOH in Coplin jars
  • 95% EtOH in Coplin jars (see recipe)
  • Ice
  • TUNEL reaction mixture (see recipe)
  • In Situ Cell Death detection Kit (Roche, cat. no. 11 684 817 910) containing:
    • Enzyme solution (TdT)
    • Label solution (fluorescein‐dUTP)
  • 300 nM DAPI working solution (see recipe)
  • ProLong Gold Antifade Mountant (Life Technologies, cat. no. P36930)
  • Slide warmer
  • Humidified chamber
  • Whatman paper
  • Cold room
  • Rocking platform
  • PAP pen
  • Incubator at 37°C
  • Glass coverslips
  • Aluminum foil
  • Microscope for fluorescence detection
  • 250‐ml Wheaton glass staining dishes (for 1 × PBS)
  • Slide racks

Basic Protocol 7: In Situ Cell Death Detection (TUNEL Assay)

  Materials
  • 5‐μm paraffin‐embedded tissue sections on Superfrost Plus slides (see protocol 1)
  • Xylene in Coplin jars
  • 100% EtOH in Coplin jars
  • 95% EtOH in Coplin jars (see recipe #5)
  • 1× Tris‐buffered saline (TBS; see recipe)
  • Sodium citrate solution (see recipe)
  • 3% hydrogen peroxide/methanol (H 2O 2/MeOH; see recipe)
  • dH 2O
  • TNB (see recipe)
  • Unconjugated primary antibody
  • Ice
  • 1× Tris/NaCl/Tween 20 (TNT; see recipe)
  • Biotin‐conjugated secondary antibody
  • DAB (3, 3’‐diaminobenzidine) Peroxidase (HRP) substrate kit (Vector Labs, cat. no. SK‐4100; see recipe)
  • TSA kit (Perkin Elmer, cat. no. NEL700A) containing:
    • Biotynil tyramide
    • Amplification diluent
    • HRP‐SA
  • Mayer's Hematoxylin
  • 1× phosphate‐buffered saline (PBS; see recipe)
  • Xylene‐based mounting medium
  • Slide warmer
  • Humidified chamber
  • Whatman paper
  • 250‐ml plastic container for sodium citrate solution
  • Water bath at 95°C
  • Kimwipes
  • PAP pen
  • Rocking platform
  • Glass coverslips
  • Slide rack
  • Slide tray
  • 250‐ml Wheaton glass staining dishes (for 1 × PBS, 1 × TBS, 3% H 2O 2/MeOH, dH 2O, TNT)

Basic Protocol 8: Immunohistochemistry on Formalin‐Fixed Paraffin‐Embedded Tissues

  Materials
  • Pregnant mouse
  • 10 mg/ml 5‐bromo‐2’‐deoxyuridine (BrdU)‐1.2 mg/ml 5‐fluoro‐2’‐deoxyuridine (FdU) solution (see recipe)
  • Ice
  • 1× phosphate‐buffered saline (PBS; see recipe)
  • 5‐μm paraffin‐embedded tissue sections on Superfrost Plus slides (see protocol 1)
  • Xylene in Coplin jars
  • 100% EtOH in Coplin jars
  • 95% EtOH in Coplin jars (see recipe)
  • 3% H 2O 2/MeOH (see recipe)
  • BrdU staining kit (Invitrogen, cat. no. 93–3943; see recipe for trypsin solution) containing:
    • Denaturing solution (Reagent #2)
    • Blocking solution (Reagent #3)
    • Biotinylated mouse anti‐BrdU (Reagent #4)
    • HRP‐streptavidin solution (Reagent #5)
    • Hematoxylin (Reagent #7)
  • dH 2O
  • DAB HRP Peroxidase substrate kit (Vector Labs, cat. no. SK‐4100; see recipe for DAB HRP substrate solution)
  • Xylene‐based mounting medium
  • Balance
  • 1‐ml syringe equipped with a 27‐G needle
  • 100‐mm petri dishes, sterile
  • Slide warmer
  • Humidified chamber
  • Whatman paper
  • Rocking platform
  • PAP pen
  • Incubator at 37°C
  • Kimwipes
  • Glass coverslips
  • Slide rack
  • 250‐ml Wheaton glass staining dishes (for 1 × PBS and dH 2O)
  • Slide tray

Basic Protocol 9: Proliferation Assays: BrdU Assay to Evaluate Cell Proliferation

  Additional Materials (also see protocol 11)
  • Streptavidin Alexa ‐Fluor‐conjugated
  • 5 mg/ml DAPI stock solution (see recipe)
  • 300 nM DAPI working solution (see recipe)
  • ProLong Gold Antifade Mountant (Life Technologies, cat. no. P36930)
  • Aluminum foil
  • Microscope for fluorescence detection

Support Protocol 2: BrdU Detection with a Fluorescent Dye

  Materials
  • Pregnant mouse
  • Click‐iT EdU Alexa Fluor 488 Imaging Kit (Invitrogen, cat. no. C10337; see recipe)
  • Ice
  • 1× phosphate‐buffered saline (PBS; see recipe)
  • 70% EtOH
  • 5‐μm paraffin‐embedded tissue sections on Superfrost Plus slides (see protocol 1)
  • Xylene in Coplin jars
  • 100% EtOH in Coplin jars
  • 95% EtOH in Coplin jars (see recipe #5)
  • Click‐iT EdU Alexa Fluor 488 Imaging Kit (Invitrogen, cat. no. C10337; see recipes)
  • Hoechst 33342 working solution (see recipe)
  • ProLong Gold Antifade Mountant (Life Technologies, cat. no. P36930)
  • Balance
  • 1‐ml syringes equipped with 27‐G needles
  • 100‐mm petri dishes, sterile
  • Slide warmer
  • Humidified chamber
  • Whatman paper
  • PAP pen
  • Kimwipes
  • Glass coverslips
  • Slide rack
  • 250‐ml Wheaton glass dishes (for 1 × PBS)

Alternate Protocol 2: EdU Assay to Evaluate Cell Proliferation

  Materials
  • 5‐μm paraffin‐embedded tissue sections on Superfrost Plus slides (see protocol 1)
  • Phosphate‐buffered saline (PBS; see recipe)
  • Xylene in Coplin jars
  • 100% EtOH in Coplin jars
  • 95% EtOH in Coplin jars (see recipe)
  • Sodium citrate solution (see recipe)
  • 3% H 2O 2/MeOH (see recipe)
  • PCNA staining kit (Invitrogen, cat. no. 93–1143) containing:
    • Blocking solution
    • Biotinylated mouse anti‐PCNA
    • HRP‐streptavidin
    • Hematoxylin
  • DAB HRP Peroxidase substrate kit (Vector Labs, cat. no. SK‐4100; see recipe for DAB HRP substrate solution)
  • dH 2O
  • Xylene‐based mounting medium
  • Slide warmer
  • Whatman paper
  • Humidified chamber
  • 250‐ml plastic container for sodium citrate solution
  • Water bath at 95°C
  • Rocking platform
  • PAP pen
  • Slide rack
  • Slide tray
  • Glass coverslips
  • 250‐ml Wheaton glass staining dishes (for 1 × PBS and 3% H 2O 2/MeOH)

Basic Protocol 10: Proliferating Cell Nuclear Antigen (PCNA) Assay

  Additional Materials (also see protocol 14)
  • Streptavidin Alexa‐Fluor‐conjugated (diluted 1:100 in blocking solution reagent #1 from Invitrogen PCNA staining kit)
  • 300 nM DAPI working solution (see recipe)
  • ProLong Gold Antifade Mountant (Life Technologies, cat. no. P36930)
  • Aluminum foil
  • Microscope for fluorescence detection

Support Protocol 3: PCNA Detection with a Fluorescent Dye

  Materials
  • Pregnant mouse
  • 10 mM EF5 stock solution (see recipe)
  • Phosphate‐buffered saline (PBS; see recipe)
  • Ice
  • 10‐μm freshly frozen tissue sections on Superfrost Plus slides (see Basic Protocol 2)
  • 100% acetone
  • 5% mouse serum/PBS (blocking solution; see recipe #60)
  • 3% BSA/PBS (antibody carrier; see recipe)
  • Anti‐EF5 conjugated antibody
  • dH 2O
  • 300 nM DAPI working solution (see recipe)
  • ProLong Gold Antifade Mountant (Life Technologies, cat. no. P36930)
  • Balance
  • 1‐ml syringes equipped with 27‐G needles
  • 100‐mm petri dishes, sterile
  • Whatman paper
  • Humidified chamber
  • PAP pen
  • Coverslips
  • Aluminum foil
  • Slide racks
  • Microscope for fluorescence detection
  • 250‐ml Wheaton glass staining dishes (for 1 × PBS and 100% Acetone)

Basic Protocol 11: EF5 Staining: A Method to Detect Hypoxia in the Developing Growth Plate

  Materials
  • DAPI image (nuclei quantification or total cell number)
  • Fluorescent signal image (positive cell quantification)
  • Image J software
  • Adobe Photoshop software

Basic Protocol 12: Quantification of Fluorescent Signal‐Labeled Cells Using Image J

  Materials
  • Brightfield image
  • Image J software

Alternate Protocol 3: Quantification of Signal‐Labeled Cells on Brightfield Pictures Using Image J

  Materials
  • Hanks’ Balanced Salt Solution (HBSS; Gibco, cat. no. 24020)
  • Mouse embryos or newborn pups
  • 70% EtOH (see recipe)
  • Ice
  • Trypsin‐EDTA 0.25% (Gibco, cat. no. 25200)
  • 10× collagenase type II stock solution (Worthington Biochemicals, cat. no. 4174)
  • DMEM, high glucose, GlutaMAX Supplement, pyruvate (Gibco, cat. no.10569)
  • Fetal bovine serum (FBS; GE healthcare, cat. no. SH30070.03)
  • Penicillin‐Streptomycin (5,000 U/ml) (Gibco, cat. no. 15070)
  • Cre Recombinase adenovirus (Vector Biolabs, cat. no. 1045]
  • β‐gal/LacZ adenovirus (Vector Biolabs, cat. no. 1080]
  • 100‐mm petri dishes
  • Dissection tools including:
    • Scalpel
    • Scissors
    • Forceps
    • Microdissecting scissors
    • Forceps
  • Stereomicroscope
  • 50‐ml tubes
  • Class II biological safety cabinet
  • Agitated water bath or oven
  • 5‐, 10‐, 25‐, and 50‐ml serological sterile pipets
  • Micropipet and sterile tips
  • 70‐μm sterile nylon mesh cell strainer (BD Falcon, cat. no. 352350)
  • Centrifuge
  • Hemacytometer
  • Primaria 6‐Well Cell Clear Flat Bottom Surface‐Modified Multiwell Culture (Corning, cat. no. 353846)
  • 37°C incubator
  • Phase contrast microscope

Basic Protocol 13: In Vitro Recombination: A Tool to Study Growth Plate Development

  Materials
  • Hanks’ Balanced Salt Solution (HBSS; Gibco, cat. no. 24020)
  • Collagenase type II (Worthington Biochemicals, cat. no. 4174)
  • 50‐ml tubes
  • Class II biological safety cabinet
  • 1‐ml syringe Luer‐Lok Tip (Becton Dickinson, cat. no. 309628)
  • 20‐G needle (Becton Dickinson, cat. no. 305175)
  • Vortex mixer
  • 1‐ml micropipets and clean tips
  • 60‐ml syringe Luer‐Lok Tip (Becton Dickinson, cat. no. 309653)
  • 18‐G needle (Becton Dickinson, cat. no. 305196)
  • 0.22‐μm syringe‐driven filter unit (Millex, cat. no. SLGV004SL)
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Literature Cited

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