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Neuronal Tracing DiI

CROCKETTDP crockettdp at aol.com
Thu Jun 22 21:19:12 EST 1995


Dear Elizabeth,

I have only recently gotten into the DiI game.  I am presently labelling
trigeminal affererent projections in baby rats (p1 - p8), in which the
alternate sections are processed for immunocytochemistry.   Basically, I
have been following the published protochols from RW Rhoades' lab  with
some modifications.  The animals are perfused using  a modification of the
pH-change method of Berod (1)a saline wash, followed by 4%
paraformaldehyde at pH 6.3, followed by (2) 4% paraform. + 15% saturated
picric acid, pH 8 and (3) 4% paraformaldehyde, pH 10.5.  I postfix the
tissue for at least a week.  Crystals of DiI are then pused into the
trigeminal ganglion with a micropipette tip.  The brains are stored at 37
degrees C for 10 - 15 days and then in the dark for an additional 3 wks.  
The results are great.   But it requires patience.

Note: I have been told that diffusion is faster in fresh tissue.  You
might try placing the crystals in an unfixed brain and then fix by
ermerison.  I have not tried this and I don't know about morphological
integrity.

D. Crockett 
David P. Crockett, Ph.D.
Department of Neuroscience and Cell Biology
UMDNJ-Robert Wood Johnson Medical School
675 Hoes Lane
Piscataway, NJ 08854



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