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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 |
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发表于 2007-8-28 00:30:01