RE: [Histonet] Help With Cutting Heart

From:"Charles Scouten"

 Sacrifice perfusion of the heart in advance would help.  Formaldehyde pentrates slowly, and fixes slowly.  Meanwhile, the tissue quality can be deteriorating.  Fixing by immersion is inferior to getting fixative to every cell through the open capillaries almost instantantly.  Try this.

Heavily anesthetize the animal, and open the chest cavity.
Enter the returning vena cava with a needle.  Cut the ascending aorta to allow outflow.  Flow 5% sucrose for a minute to wash out blood and extracellular  sodium.  Flow PFA for 5 mintues or more to thoroughly penetrate tissue.  Let sit for 4 hours, with very slow flow, or no flow, of fixative.  Flow 30% sucrose for 1 minute to thoroughly penetrate throughout the muscle.  For immunology where overfixation can be a problem, this is enough.  If for Nissl or other simple stain, add some PFA to the 30% sucrose.  Remove heart, and let sit in 30% sucrose (with or without fixative, depending on intended use) for a day or so. It will already be cryoprotected and throughly penetrated with sucrose, so 5 days is unnecessary.  

An instrument to readily handle the fluids can be found at:

Charles W.  Scouten, Ph.D. 
5918 Evergreen Blvd. 
St. Louis, MO 63134 
Ph: 314 522 0300  
FAX  314 522 0377 

-----Original Message-----
From: [] On Behalf Of
Sent: Wednesday, April 28, 2004 10:52 AM
Subject: [Histonet] Help With Cutting Heart

Hello Histonetters,
   I have been cutting transverse sections of rat heart using the following protocol:
1) Dissect heart, cut in 3mm disks
2) Fix overnight 4% PFA @ 4 deg.
3) Cryoprotect in 30% sucrose around 5 days.
4) Embed in OCT and freeze blocks in isopentane that has been
   cooled in liqid N2 for 1min.
5) Store 6 hours at -80 deg.
6) Store o.n. at -20 deg and cut 16uM on cryostat also at this
   The quality of the resulting histology is o.k but not great, specifically the endocardium is a bit torn.   I cannot parraffin embed because of downstream in-situ hybridization signal intensity problems so i must use a frozen or perhaps fresh tissue histology protocol. Does anyone have any suggestions or experience with good frozen heart histology, any changes i could make to my protocol? maybe a new protocol? Please help! 
Thanks in advance,
Fred Grau
Dept. Neurbiology & Behavior
SUNY Stony Brook

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