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| Beta-Gal staining in primary neurons
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| [ 文章来源： | 文章作者：
| 发布时间：2007-01-28|
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Beta-Gal staining in primary neurons
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Fix cells w/ 4 ml fix soln/10cm or 60 cm dish, 37 C, 5 min
Fix soln.: for 100 ml (use w/i 1 wk)
5.4 ml 37 % formaldehyde (Final 2%)
800 ul 25% Glutaraldehyde (fin. 0.2%)
94.5 ml PBS
- Wash cells 1x w/ PBS prewarmed to 37 C
- Stain w/ 2 ml staining soln./60 mm plate for 30 min to o/n. Blue cells may start to appear in 30 min.
Stain soln.: part a. 5 mM Potassium Ferricyamide (K3Fe(CN)6)
5 mM potassium ferrocyamide (K4Fe(CN)6.3H2O)
2 mM MgCl2
in PBS. Store @ R.T., use w/i 1 month or until see ppt.
For 200 ml of a. : 0.33 g K3Fe(CN)6
0.42 g K4Fe(CN)6.3H2O
0.4 ml 1M MgCl2
Part b. X-gal (5-bromo-4-chloro-3-indolyl-beta-gal)
40 mg/ml in DMSO
Heat part a & b to 37 C
Add part b to part a to final 1 mg/ml X-gal. Add part b slowly while mixing, or crystals will form.
Filter to remove crystals.
- Remove stain soln, either replace w/ fixer & store @ 4 C, or wash w/ PBS and mount.
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