[Histonet] vibratome sections for cavalieri volume estimation

[Claudia Lutz]

Hi,

I am fairly new to histological procedures and new to this list.  My lab
would like to stain vibratome sections of honey bee brain with phalloidin
in order to estimate volume of specific brain regions.  I am fixing the
brains overnight in 4% paraformaldehyde, embedding in agarose, sectioning
at 100 um, then staining free-floating sections in phalloidin with PBS
with 0.2% Triton-X.  I am mounting the slices in 80% glycerol and then
imaging with the confocal microscope.  I would like to use stereological
procedures (specifically the cavalieri volume estimator) to quantify the
volume of specific brain regions using systematic sampling of 10um optical
sections that I obtain from the slices.  I have several (possibly naive)
questions:

1. My 100 um sections are shrinking to 60 um (or less) by the time I image
them, judging by where I find or lose focus while imaging.  What can I do
to minimize this?  How can I properly quantify the degree of shrinkage, to
see whether or not it is uniform?  Can I avoid bias in my volume
estimation?

2. My phalloidin staining appears much brighter in the center of the
slice, even when I use settings to adjust the gain while taking z-stacks. 
Does this indicate tissue damage at the edges?  Can I ignore this?  This
also relates to my question above, since I am not sure I can precisely
find the edge of the slice by focusing when I need to increase the gain.

3. I lose order and orientation of the slices because I am staining them
free-floating.  Does anyone have experience staining 40 or 50 um vibratome
slices on the slide with phalloidin or antibody, or have a trustworthy
protocol for this?

Sorry to ask so many questions, and thank you for any help.  There is not
a lot of expertise in my lab on this, so I am feeling frustrated.

Claudia Lutz
University of Illinois at Urbana-Champaign
Neuroscience Graduate Program