Protocols
Xgal staining
Frozen Section Xgal Stain
1. Cut unfixed frozen sections. Air-drying is not harmful to Bgal activity.
2. Fix 0.5% Glutaraldehyde in PBS for 1 minute. PFA fixation kills enzyme activity.
3. Wash with PBS 10 minutes.
4. Place slides in humidified chamber
5. Add Xgal solution (premade as per wholemount protocol), important that pH is not lower than 7.5 or endogenous Bgal activity will be high.
Xgal Solution:
5mM K3Fe(CN)6,
5mM K4Fe(CN)6,
2mM MgCl2,
0.01% Na deoxycholate,
0.02% NP-40,
1mg/ml Xgal,
Make up at least 24 hrs before, decant before using to avoid crystals on sections.
6. Incubate at 37 degrees for several hours, or overnight at RT or 4 degrees
7. Monitor, stop by washing in PBS
8. Counter stain 1-2 minutes nuclear fast red, Eosin works well too.
9. Wash ddH20 several times, then mount in crystalmount and then permount.
RFP Detection