[Histonet] frozen tissue

[Patsy Ruegg]

I am having trouble getting tissue to cut that are prepared as such:

"Animals were heparinized through the inferior vena cava under Avertin
anesthesia.  Hearts were removed, cannulated through the aorta, and reverse
perfused with cardioplegia solution (physiological buffer containing high
concentrations of KCl and EGTA) for 5 min at 2 ml/min to clear blood and
fully relax the heart.  Perfusion was then switched to 4% paraformaldehyde
in PBS for 5 min.  Hearts were removed from the perfusion apparatus,
ventricles were dissected free of aorta and connective tissue, and hearts
were stored overnight at 4C in cryoprotectant solution (0.1 M phosphate
buffer, 30% sucrose, 30% ethylene glycol)."

I took these tissues and snap froze them in liquid nitrogen in cryomolds
filled with OCT as I usually do frozen tissue, as I tried to cut them in the
cryostat they seemed raw as if the glycol had effected the freezing, the OCT
surrounding the tissue was well frozen but the tissue seemed not so well
frozen.

Does anyone have experience with freezing tissues that have been place in
ethylene glycol cryo protectant, I have not seen this before.

Any advice would be appreciated.  Some of these tissues are still in 30%
sucrose which I transferred them to to try and rinse out the glycol.

Thanks,

Patsy

 

 

Patsy Ruegg, HT(ASCP)QIHC

IHCtech, LLC

12635 Montview Blvd. Ste.215

Aurora, Colorado 80045

Phone: 720-859-4060

Fax: 720-859-4110

pruegg <@t> ihctech.net

www.ihctech.net 

www.ihcrg.org