GFP骨的冰冻切片实验方法

Protocol for Frozen section of GFP Bone:

1. 1. Fix the bone in 4% Paraformaldehyde at 4�C under constant agitation for 3 days.

   2. Decalcification in 14% EDTA solution at 4�C or RT under constant agitation for 3~5 days (change fresh 14% EDTA solution every 24 hours).

   3. Wash in PBS for 2 hours.

   4. Soak in 30% Sucrose in PBS at 4�C under constant agitation overnight.

   5. Embed bone in OCT compound.

   6. Cut 5µm thick frozen sections using cryostat and allow the sections to dry ( keep in 
      dark).

   7. Rinse in PBS 5min x 2.

   8. Soak in 1mM MgCl2 in PBS for 30min.

   9. Rinse in PBS 5min.

   10. Mount coverslide using 50% Glycerol in PBS.

   11. Examine by fluorescence microscopy.

Solution: 

4% Paraformaldehyde in 0.1M PBS:

• Heat PBS to ~ 65�C to dissolve Paraformaldehyde,then cool it and adjust pH to 7.4. ( use fresh solution)

Decalcification solution:

• 280 g EDTA

• ddwater 1.5 L

• 180 ml Ammonium Hydroxide

• Mix well and adjust pH to 7.1 with Ammonium Hydroxide

• Add water to 2 L.