I am soliciting opinions/advice about floating section ICC in mouse brain. i
know this is probably pretty basic stuff, but any help will be greatly
The brains are fixed in 4% para, then cryoprotected with 20%, then 30%
I am trying to embed in gelatin rather than TBS/OCT because of the added
control it gives in orienting the brain, the opportunity to put a side
marker in the gelatin around the slice, and also because it is sometimes
helpful in keeping potential "float away" regions (e.g. cortex in more
posterior portions, mammilary nucleus...)
connected to the rest of the brain.
1) Any suggestions about the ideal gelatin percentage? Should I add sucrose
to get the gelatin to adhere better to the brain--or will that make the
sections sticky and hard to handle. Is post fixing the gelatin (after the
brain is embedded) a good idea? Should it then be rinsed in PBS for a while
to get rid of excess fix?
2) In our first try at this using 15% gelatin, I noticed the method of
freezing that we used for TBS/OCT--freezing in a cryomold imobilized in a
dry ice ethanol slurry/snow did not seem to work well. The tissue was full
of holes. Is this coincidence or is it because the solidified gelatin block
is a poor heat conductor? If that is the case, should I just drop the block
into isopentane/dry ice, or even a dry ice ethanol bath (instead of
3) I need hints about getting the floating section on the slide at the end
of the experiment in a quicker, more efficient manner. Thus far we have
found some Triton-X 100 in the PBS to be helpful. Is there an ideal
concentration? Are other additives helpful? Should we be using something
other then Fisher Superfrost Plus slides? Any special brush or droplet
techniques compatible with multiple sections on a slide?
Thanks in advance for any help with these questions!
Department of Psychiatry
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