[Histonet] 1uM thick Drosophila sections

[Danielle Crippen]

Dear Experts in HistoLand,

I need to make 1uM thick sections of full grown Drosophila for Toluidine Blue staining and have some questions about fixation and processing.

1.) 	What is the best way to get the fixative into the fly?  I've read several techniques ranging from careful incision into 	abdomen to cutting off the head?  Secondarily--is the best tool for this a microscalpel, or can other instruments be 	employed?
2.) 	What is the best fixative?  We're not doing EM, just need thick sections from the ultramicrotome.  Does the 	standard 2% PFA/ 0.2% gluteraldehyde in CB suffice?
3.)	I've read that some fix/process under vacuum?  How common/necessary is this?  And--how long is the fixation 	time?
4.) 	I've also read that some put 1%DMSO into all reagents through absolute alcohol.  Is this standard?

A thousand thanks in advance!!

Cheers,

Danielle