GFP骨的冰冻切片实验方法
Protocol for Frozen section of GFP Bone:
Fix the bone in 4% Paraformaldehyde at 4�C under constant agitation for 3 days.
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).
Wash in PBS for 2 hours.
Soak in 30% Sucrose in PBS at 4�C under constant agitation overnight.
Embed bone in OCT compound.
Cut 5µm thick frozen sections using cryostat and allow the sections to dry ( keep in
dark).Rinse in PBS 5min x 2.
Soak in 1mM MgCl2 in PBS for 30min.
Rinse in PBS 5min.
Mount coverslide using 50% Glycerol in PBS.
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.
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