[Histonet] A flurry of questions about gelatin embedding, freezing, and mounting for mouse brain floating sections.

[Joshua Berman]

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 
appreciated.

The brains are fixed in 4% para, then cryoprotected with 20%, then 30% 
sucrose.
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 
snow/slurry).

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!


Joshua Berman
Department of Psychiatry
Columbia University