[Histonet] Mice embryonic brain - EM Poor tissue quality - need help

[Davis, Gareth]

I'm not sure about EM procedures, but I've been told, and do find, perfusion fixation to be the best for fixing brain tissue.  If you need a protocol for that I have one.
Gareth
 
Ms. Gareth B. Davis
Research Assistant II
Neuro-magnetics Division of 
the Department of Neurology
Vanderbilt University Medical Center
615-936-3318
 
 

	-----Original Message----- 
	From: Rowani [mailto:rowani.mohdrawi <@t> student.adelaide.edu.au] 
	Sent: Mon 9/15/2003 12:08 AM 
	To: histonet <@t> pathology.swmed.edu 
	Cc: 
	Subject: [Histonet] Mice embryonic brain - EM Poor tissue quality - need help
	
	

	Hi
	My electron microscopy sections of brain of mice foetuses (18.5 dpc)  are
	difficult to interpret because of  disrupted and lysed cells. The connective
	tissue surrounding the cells  appears disintegrated with empty
	spaces/vacuoles like artifact ?.  The brain was processed according to the
	following protocols: brain was dissected and immersed in ice cold fixative
	(glutaryldehyde 2.5%, paraformaldehyde 4%, sucrose 2% in PBS pH 7.2) for 1-2
	hrs to firm it up before cutting them to smaller fragments (2 cumm). After
	an overnight fixation, re-fixed in osmium tetroxide, washed in PBS,
	dehydrated through a series of ethanol (70%, 90%, 95% and 100%) and embedded
	in araldyte resin.  The brain of adult mice processed using similiar
	protocol was excellent.  COuld someone suggest ways to improve the tissue
	quality ?
	
	TQ
	
	Rowani
	
	
	
	
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