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
    The more dehydrated the tissue is before adding BABB the better

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)

Hama H, Kurokawa H, Kawano H, Ando R, Shimogori T, Noda H, Fukami K, Sakaue-Sawano A, Miyawaki A. 2011. Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain. Nat Neurosci. 14:1481-8.   |  Overview in Biotechniques

Ertürk, A., C.P. Mauch, F. Hellal, F. Förstner, T. Keck, K. Becker, N. Jährling, H. Steffens, M. Richter, et al. 2011. Three-dimensional imaging of the unsectioned adult spinal cord to assess axon regeneration and glial responses after injury. Nature Medicine 18:166-171.   |  Overview in Biotechniques 1

Citifluor  |  TDE  |  Tetrahydrofuran 1

		Copyright 2001-2014  Dr. M. Chua, Program in Molecular Biology & Biotechnology, School of Medicine, University of North Carolina, Chapel Hill, NC 27599
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