骨组织β-gal免疫染色实验技术方法

互联网2013-09-06

6798

ß-gal Staining:
Reagents:
	0.1M Phosphate Buffer (pH 7.3):
	0.1M Sodium Phosphate monobasic	115ml
	0.1M sodium phosphate dibasic	385ml
	Total Volume	500 ml

Fix solution:
	0.5% Gluteraldehyde:
	25% gluteraldehyde	0.4 ml
	100mM EGTA pH 7.3	2.5 ml
	1M magnesium chloride	0.1 ml
	0.1 M sodium phosphate pH 7.3	47.0 ml
	Total Volume	50.0 ml

	4% Paraformadehyde:
	paraformadehyde	20 g
	1M magnesium chloride	1ml
	100mM EGTA	25ml
	~500 ml PBS
Prepare fresh each time

Wash buffer:
	1M magnesium chloride	0.4 ml
	1% deoxycholate	2.0 ml
	2% Nonidet-P40	2.0 ml
	0.1M sodium phosphate pH 7.3	195.6 ml
	Total Volume	200.0 ml

X-gal Staining:
	25mg/ml x-gal stock dissolved in di-methyl formamide	2.0 ml
	potassium ferrocyanide (Sigma P-9387)	0.106 g
	potassium ferricyanide (Sigma P-8131)	0.082 g
	wash buffer	48.0 ml
	Total Volume	50 ml
This buffer can be reused, filter after use and store in the dark.
Also note, crystal from due to di-methyl formamide. If these crystal are a problem, prepare X-gal stock in dimethyl sulfoxide.

X-gal Staining of Bone:

Whole mount staining:

Dissect bone out of mice.

Fix at tissue in fixative for 30min-2h at RT or overnight in 4�C with 4% paraformadehyde.

Rinse with PBS.

Soak tissue in X-gal staining solution for 4 hours at 37�C.

Pour off the staining solution, replace with wash buffer, 2 times.

Sock tissue in 30% sucrose in PBS.

Embed and cut frozen section.

Frozen section staining:

Bone after fixed and briefly rinse with PBS.

Soak with decal solution. Gently rock at 4�C for 1-3 days.

Soak with 30% sucrose in PBS + 2mM MgCl2 at 4�C for overnight.

Embed in OCT.

Cut frozen section. Place slides in wash solution.

Incubate sections with x-gal solution at 37�C for 1-3 hours (monitor staining every 30 min. It usually takes 2 hours).

Rinse slides in PBS, 3 times.

Rinse with 95% EtoH and stain with Eosin 20 secs for counterstain.

Dehydrate and coverslip