| |||
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 | |||
| |||
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 | ||
| |||
| |||
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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