与骨骼相关的组织学实验技术方法

Embedding Bones for Frozen Section:

1. Fill an ice bucket with dry ice pellets and place a 500ml beaker in the middle.

2. Put approximately 200ml of 2-MethylButane in the beaker and cover the bucket. (The MethylButane will get extremely cold, allowing you to ‘flash freeze’ your samples).

3. Label your molds and fill the molds with Frozen Embedding Medium (Thermo Shandon, Pittsburgh, PA).

4. Immerse bone in embedding medium and adjust right position as needed.

5. Test 2-MethylButane with dry ice pellets, if it does not boil, it is ready.

6. Use a forceps to keep the embedding mold horizontaly for few seconds until the embedding medium is frozen in pre-cold Methybutane and allow it to sit to the bottom of the beaker for at least 2 minutes to ensure it is frozen all the way through.

7. Frozen samples can be placed on dry ice in the bucket until all samples are done. Wrap your samples in aluminiumfoil.

8. Place in a sealed container and store your samples at -20�C or -80�C.

Trap Stain:

Staining Procedure:

1. Preheat to 37°C 2 Coplin jars with 50ml of Buffer I Solution each.

2. Take one Coplin jar and add 0.5 ml of Buffer II Solution, add slides and incubate at 37°C for 45 minus.

3. A few min before the time is up, mix 1 ml of Buffer III Solution and 1 ml of Buffer IV solution for 30 sec and let it sit for 2 min.

4. Add this mixed solution to the other Coplin jar of 50 ml Buffer I Solution, mix and add the slides without rinsing.

5. Incubate at room temp ~5 min.

6. Rinse, counterstain with hematoxylin for 40 sec and put slides in 0.05% Ammonia water.

7. Dehydrate through alcohol, clear in xylene and mount.

Alizarin Red S Staining - Calcium
Alizarin Red S Solution:
Alizarin Red S	2 gm
Distilled Water	100 ml
Mix the solution and adjust the pH to 4.1-4.3 using 0.5% ammonium hydroxide. The pH is critical - make fresh.
Acetone – Xylene:
Acetone	25 ml
Xylene	25 ml
Make fresh.

Procedure:

1. Rinse slides rapidly with distilled water.

2. Alizarin red S solution, 30 sec to 3 min, checking microscopically for the orange-red color.

3. Shake off excess dye.

4. Acetone:- 20 dips.

5. Acetone-xylene:- 20 dips.

6. Clear in xylene, mount in permount.

Goldner Trichrome Staining
Weigert Hematoxyline:
	Solution A
	Hematoxyline	5 g
	95% Alcohol	500 ml
	Solution B	5 g
	Ferric chloride	5.8 gl
	Deionized water	500 ml
	Concentrated Chlorhydric Acide	5.0 ml
24 hours before use, mix solutions A and B in equal amounts  Stable for 8 days.
	Fushine-Ponceau:
	Fushine acid	0.167 g
	Ponceau S	0.667 g
	Deionized water	500 ml
	Acetic Acid (glacial)	1.0 ml
Shake well and filter.
	Orange G:
	Phospho-molybdic Acid	25 g
	Deionized water (Shake well)	500 ml
	Add Orange G	10 g
Shake well and filter.
	Light Green:
	Light green	1.5 g
	Deionized water	500 ml
	Acetic Acid (glacial)	1 ml
Shake well and filter.
	Acetic Acid 1%

Staining Procedure:

1. Put slides in deionized water.

2. Mordant in Bouin’s Fixative (Poly Scientific Inc. Catalog# s129-16oz) at room temperature overnight.

3. Rinse with deionized water frequently for 10 minutes.

4. Weight Hematoxyline 15 minutes.

5. Rinse with deionized water frequently for 10 minutes.

6. Fushine-ponceau 30 minutes.

7. Rinse with 1% acetic acid rapidly.

8. Orange G 8 minutes.

9. Rinse with 1% acetic acid rapidly.

10. Light Green 20 minutes

11. Rinse with 1% acetic acid rapidly.

12. Dehydrate through alcohol, clear in xylene and mount.