Michael Hooker Microscopy Facility (MHMF.ORG)

Clearing Agents / Procedures

Dehydrated it in 70, 95 and 100% EtOH, then methyl salycilate

BABB (Sigma, St. Louis, MO) for clearing tissue - benzyl alcohol and benzyl benzoate. RI is 1.54-1.57

Murray's Clear - 33% Benzyl Alcohol + 67% Benzyl Benzoate

Glycerol Mounting Medium with an antifade

• 0.3 M PBS (2X)  q.v. 10 ml
• 5.7 g/10ml Glycerol
• 0.25 g/10ml N-Propyl Gallate
• Stir overnight

DABCO (Giloh and Sedat; Science, 1982)

• 1% DABCO

• 10% PBS

• 90% Glycerol

• 4% N-Propyl Gallate

• (1) Dissolve in beaker (0.5 L for 100 mL solution):
•  DABCO (1,4-Diazabicyclo[2.2.2]-octane, Aldrich) 2.5% (w/v) and Mowiol 4-88 (Calbiochem) 10% (w/v)  in 0.1M TRIS-HCl, pH 8.5 and 25% (w/v) glycerol
   
• (2) Heated magnetic stirrer at low speed, about 50 C.
   
• (3) After all is dissolved, aliquot 0.25 mL into microtubes, store -20 C in the dark (good for well over one year)
   
• (4) Before use, heat to 65 C for 10 min, let cool 1-2 min and use immediately on specimen. Discard remains. This mounting media will solidify overnight in the refrigerator (4 C, dark), but is not as hard as epoxy mounts, so handle gently.
   
• (5) I measured the refractive index as R=1.4101 @ 22.0 C (water 1.33, oil 1.56)
   

		Copyright 2001-10  Dr. M. Chua, Office of Research, School of Medicine, University of North Carolina, Chapel Hill, NC 27599
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