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Jim

The neurons were in sympathetic ganglia and all the tissue was treated
as whole mounts. The ganglia are several hundred microns thick, but the
neurons of interest were within about 100 microns of the top surface.
We measured them directly in the X-Y plane, since it was a bit hard to
get exact z-axis info during the different processing steps due to
different lens and focussing systems etc. So whatever else was going
on, I don't think the glass was providing much lateral support.

A slightly more scary observation we've made a few times is that
cryostat sections apparently firmly stuck to slides can show distortion
if they are dehydrated and mounted in DePeX. I have no idea how or why
this happens, but it was one of the reasons we switched to using PEG
embedding whenever we can (this process also helps with minimal
dimensional change)

Cheers

IAN

On Wednesday, December 3, 2003, at 12:31  PM, James Pawley wrote:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>> Search the CONFOCAL archive at
>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>
>> Jim, Mike and listers,
>>
>> I suppose one conclusion from this is not to dehydrate your tissue!
>> Also, preparation for SEM causes more shrinkage than many
>> procedures, primarily as a result of the critical point drying step,
>> as I recall. (c.f. For TEM, shrinkage of the polymerising resin
>> seems to be pretty important).
>>
>> For our routine immunohistochemistry, we have only very limited
>> exposure to solvents for permeabilising membranes (ethanol and DMSO)
>> and we mount in buffered 50% glycerol). I think I said this on the
>> list before, but a few years ago we tracked the dimensional changes
>> of dye filled neurons right through from when they were alive until
>> after all the processing steps, and found very little change under
>> our standard protocols (see J Neurophys 86: 1237-1251, 2001).
>>
>> hope that helps
>>
>> IAN
>>
>
>
> Actually, Boyde and Maconnachie found that most of the shrinkage was
> in the dehydration step even with freeze dried specimens, although
> fixation also caused some.
>
> I echo Ian's idea about not dehydrating if size is important. And
> glycerol is probably the next best thing
>
> I would also be interested to know a little more about the neuron
> study mentioned above: where these 3D measurements or 2D (in plane)
> measurements? Glass is quite stiff and tissue adhering to it is often
> stabilized in the xy plane.
>
> Cheers,
>
> Jim P.
> --
>               ****************************************
> Prof. James B. Pawley,                             Ph.  608-263-3147
> Room 223, Zoology Research Building,               FAX  608-265-5315
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>
>

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Prof Ian Gibbins
Anatomy & Histology
Flinders University
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