Histomorphometry on unon-decalcified bone samples

Sample preparation

 

  1. Inject mice intraperitoneally with two fluorochrome labels at set intervals of time: 1 month-old mice with 20 mg/kg tetracycline 3 days before sacrifice, and with 10 mg/kg calcein 1 day before sacrifice; 2 month-old mice with 20 mg/kg tetracycline 4 days before sacrifice, and with 10 mg/kg calcein 1 day before sacrifice.
  2. Shortly before use, make a 5 mg/ml solution of tetracycline or a 2.5 mg/ml solution of calcein in 2% w/v NaHCO3 pH 7.4. Filter and inject intraperitoneally 4μl/g weight.
  3. Sacrifice at 24 hours after the last injection. Excise the femora immediately after sacrifice and clean from the surrounding soft tissues.
  4. Fix and store bone specimens in 70%EtOH.

 

MMA preparation

  1. Put 1 L of commercial MMA in a 2 L separatory funnel. Add 200 mL of 0.5 N NaOH, mix well and wait until the two phases are separated. Open the tap and release by gravitation the lower, darkest phase, which contains the hydroquinone bound to NaOH. Repeat the operation twice. Transfer the washed MMA in a glass bottle and store it at -20°C.
  2. Filter MMA in order to discard water crystals. The MMA washed and filtered can be used to prepare the working solution.
    Solution A: Dissolve 5.5 g of Benzoyl Peroxide in 500 mL of MMA washed and filtered, then add 56.1 mL of Polyethylene Glycol 400. Store in glass bottles at -20 °C. To avoid polymerisation, fill bottles half.
    Solution B: Dissolve 1 mL of N,N-Dimethylaniline in 19 mL of isopropanol. Store at 4 °C.
    Working solution: Mix Solution A and Solution B in a 50:1 ratio. Make fresh shortly before use!



Notes

  1. Always work under a fume hood when using MMA
  2. All the glassware that has been in contact with MMA needs to be cleaned in 70% EtOH before washing.

 

Block preparation

70% EtOH 15 min at 4 °C

               ↓
90% EtOH 2×1h at 4 °C
               ↓
100% EtOH 2×1h at 4 °C
               ↓
transfer samples to small glass bottles
               ↓
xylene 3×15 min at room temperature
               ↓
MMA working solution 2×3 days at -20 °C
(fill glass bottles half way to avoid polymerisation and close them with an airtight lid)
               ↓
MMA working solution 1 day at 4 °C
(fill glass bottles full to allow polymerisation, close them with a new airtight lid and parafilm)
               ↓
break the glass bottles, trim blocks and store them at room temperature

 

Sectioning

Sagittal sections (5 or 12μm thick) of the central region of the distal femur are cut parallel to the long axis of the bone, using a microtome with a tungsten carbide knife. Mount sections on gelatin-coated slides, flattened with the help of a drop of 30% EtOH, cover with a plastic coverslip and dry. Stack the slides in piles and store for at least 3 days under pressure.

 

Microscopy

Fluorescence microscopy: leave 12μm thick sections unstained, wash quickly in xylene and mount using a synthetic mounting medium (Entellan or similar).
Light microscopy: deplastify 5μm thick sections with 2-methoxyethyl acetate before staining with Masson’s Trichrome (modified), Toluidine Blue, or TRAP, according to standard
procedures.

 

Deplastification protocol

2-methoxyethyl acetate 3×15 min
               ↓
100% EtOH 2×2 min
               ↓
70% EtOH 2 min
               ↓
40% EtOH 2 min
               ↓
distilled water 2×2 min

 

Modified Masson's Trichrome protocol

Fuchsin Ponceau solution (0.5 g of Acid Fuchsin and 0.5 g of Ponceau 2R in 100 mL of 1% Acetic Acid, stored at 4 °C, equilibrated to room temperature and filtered (0.45μm) before use) 2 min
               ↓
1% Acetic Acid 1 min
               ↓
Distilled water 1 min
               ↓
Aniline blue solution ( 2.5 g of Aniline Blue in 100mL of double distilled water with 2.5mL of glacial Acetic Acid, stored at 4 °C, equilibrated to room temperature and filtered (0.45μm) before use)15 min
               ↓
Clean the back of the slides with some paper and let them dry for at least 1 hour
               ↓
Wash quickly in xylene and mount with a synthetic mounting medium (Entellan or similar)

 

Toluidine Blue staining protocol

1% Toluidine Blue (adjust pH to 4.3 with NaOH, store at 4 °C, equilibrate at room temperature and filter (0.45μm) before use) solution in BT buffer (0.63 g of Citric Acid and 0.30 g of Disodium Phosphate in 400mL of double distilled water,pH 8.0).15 min
               ↓
wash in tap water
               ↓
let dry for at least 1 hour
               ↓
tert-butyl alcohol 2×15 sec
               ↓
tert-butyl alcohol : Xylene (1:1) 15 sec
               ↓
xylene 3×15 sec
               ↓
let dry for at least 1 hour and mount with a synthetic mounting medium (Entellan or similar)