与骨骼相关的组织学实验技术方法

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.