A 1:2 mixture of benzyl alcohol : benzylbenzoate (both available from Sigma)
was originally developed by Andrew Murray to clear 1.2 mm diameter Xenopus
eggs/embryos (by matching the refractive index of yolk) (see Klymkowsky and
Hanken, Methods in Cell Biol. 36, 419; 1991; Gard, Methods in Cell Biol. 38,
241; 1993; Gard and Kropf, Methods in Cell Biol. 37, 1993). We are able to
serially optically section oocytes as large as 150 um. With larger samples,
sectioning is limited by antibody penetration and objective working
distance. This clearing solution has also proven useful for clearing a wide
variety of plant and animal tissues. Caveats:
 
The sample must be fully dehydrated prior to clearing (methanol, ethanol, or
acetone all are sufficient).
 
Sealing coverslips can be a bit of a problem. Some sealants are miscible
with the clearing solution. We use Sally Hanson's clear fingernail polish
"Hard as Nails".
 
The clearing solution dissolves some plastics.
 
 
>Hi, Everyone,
>
>I am using confocal to scan through very thick nervous tissue (ca. 150um).
>The
>problem is that when I scan deeper in the tissue, I start to loose signal.
>So two questions related:
>
>1)      What I did was to gradually increase the gain of photomultiplier to
>compensate for decreasing of the signal which makes the image grainy (though
>I can live with it). Any tips for a better way to solve the problem?
>
>2)      I tried several ways of clearing tissue. Obviously all the aqueous
>mounting medium does not clear well (or at all, including Vectasheild,
>Glycerol
>-gelatin). So far, the one works best is to dehydrate the tissue first and
>using Methyl Salicylate as clear agent. Any other mounting medium or clearing
>agent works better?
>
>        I use a 25X immersion lens (NA=0.8) or a 40X oil lens (N.A.=1.3) and
>a Zeiss LSM 410 with a Krypton/Argon laser. Most of the time I use Propidium
>iodide, Cy3 as fluorophore.
>
>Any comment is welcome. You can send me the comments directly (Email address
>is
>by the end of this message) if you believe it is not commonly interesting.
>TIA.
>
>                                                Xue Jun Sun
>
>Xue Jun Sun, Ph.D.
>Dept. of Psychology
>Dalhousie University
>Halifax, N.S. B3H 4J1
>Canada
>Fax: (902) 494 6585
>Phone (902) 494 3746
>
>Email: [log in to unmask]
>
>
David L. Gard
Assoc. Professor
Dept. of Biology
University of Utah
Salt Lake City, UT 84112
T: 801-581-7365
F: 801-581-4668
E: [log in to unmask]