[Histonet] neuronal recontruction with polar tracer/ tissue clearing with glycerol / shrinkage issue

[Sebastien Thuault]

Hi,

Does anyone have experience with glycerol clearing? I am trying to clear
very thick slices of brain tissue fixed in PFA  (400 um) but do not want to
use ethanol + xylene as I find the procedure makes the tissue shrink. I
inject biocytin in one or two neurones and then reaveal morphology with a
streptavidin coupled to peroxydase +  DAB/H2O2 procedure. When I air dry the
tissue and use ethanol and xylene, the slice shrinks a lot and the neuron's
dendrites tends to become very sinuous. I find that using glycerol
(increasing concentration from 25% to 100% in15% increments, 60 min in each
bath) just after the DAB step prevents the distortion but is not as good as
xylene to clear the tissue and very time consuming. Does anyone know of an
alternative to limit tissue distortion/shrinkage and/or clearing? I f
someone using the same morphological reconstruction technique has experience
with this problem or has a protocol that does not lead to tissue distortion
please post!

Thanks!

Sebastien Thuault, PhD
Center for Neurobiology and Behavior
Columbia University
PI Annex/Kolb Institute
1051 Riverside Drive
New York, NY 10032

Phone: +1-212-543-5037
Fax: +1-212-795-7997