[Histonet] A question on setting up of organotypic cultures , on the use of heparin in perfusion fluid (non-fixative) and finally , sucrose and ECF

From:"Hanna-Mitchell, Ann"

Good Evening from a very wet and windy Ireland!
I am setting up  spinal cord slice cultures  . 
Does anyone have any particular opinion on the use of the anti-coagulant
heparin in perfusion buffer ( non-fixative)prior to removal of 'wet' tissue
( in my case spinal cord)  to be processed for culture. It is used by some
researchers in my Department but in this instance they are pre-perfusing
with 'buffer' prior to perfusing with fixative for other purposes. 
As I want to maintain the tissue in as healthy a condition as possible, I
worry that heparin in my protocol  might have unwanted effects on it .  At
the moment I have stopped using heparin.

As regards sucrose, how does pressure perfusion cause sucrose molecules to
move from the intravascular to the extravascular compartment in the brain?  

Lastly,I should really appreciate some advice on the following: 
having sliced the spinal cord segment  on a tissue chopper (slice thickness:
400Ám),and having separated out single slices , how does one get these
slices onto a millicell insert?

Thank you for reading this . I should very much appreciate any assistance

Yours Sincerely,
Ann T. Hanna-Mitchell PhD

Dr. Ann T. Hanna-Mitchell  
Department of Anatomy
BioSciences  Institute
University College 

Phone +353 21 4902246/+353 21 4901309
E mail a.hannamitchell@ucc.ie/athm@ucc.ie


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