[Histonet] Frozen section problems!!
We are wanting to section peripheral gang= lia at 10um using a
cryostat. Our current procedure is as follows:
1)perfused with PBS/8% Para, ganglia tissue removed
<= FONT face="Default Sans Serif,Verdana,Arial,Helvetica,sans-serif"
color= =#000000 size=4>2)tissue rinsed in PBS under agitation for
3)placed in 30% sucrose overnight (or until= tissue sinks)
4)rinsed in PBS/dH20
5)embedded in OCT
6)frozen rapidly in liquid nitrogen
7)tissue allowed to equilibrate to cryostat temp & sectio= ned in
cryostat (-18 C)
8)sections= are picked up with room temp slides and dried at RT for an
9)Slides are dipped in clearing agent (xylenes)= followed by graded
alcohol (95%, 70%, 50%), dH2O, cresyl violet stain, dH2= 0, 50%, 70%,
95%, & xylenes...and coverslipped immediately.
The problem we are having is that the tissue looks outstanding after sectioning as long as it remains wet (i.e., when we take a look at
tissue d= uring the cresyl violet stage above)--once it begins to dry
we're losing ce= lls and overall tissue integrity declines rapidly--It
seems that the dehydr= ation steps following the staining of the
tissue is the culprit. We have tr= ied several modifications of this
procedure but have yet to find a successf= ul way to prevent damage of
the tissue. If you have any suggestions p= lease let me know. This
problem has been detrimental to our work for the pa= st 3 months.
University of Nebraska at Om= aha
Department of Psychology
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