Beta-Gal staining in primary neurons

Steven Finkbeiner, Departments of Neurology and Physiology, UCSF

http://gweb1.ucsf.edu/labs/finkbeiner/Protocol/protocol_list/beta-gal_staining.shtml

1. 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
   
   

2. Wash cells 1x w/ PBS prewarmed to 37 C

3. 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.
   
   

4. Remove stain soln, either replace w/ fixer & store @ 4 C, or wash w/ PBS and mount.