Re: [Histonet] Help With Cutting Heart
While it might be possible to perfuse the heart with the method
Charles describes below, the perfusate will take a rather lengthy course
to get to the heart muscle. If one perfuses via the vena cava (either
inferior or superior) the perfusate will enter the r. atrium then the r.
ventricle, the pulmonary trunk, the pulmonary arteries, pass through the
vascular beds of the lungs, enter the l. artium, the l. ventricle, out
the aorta and finally to the coronary arteries. True, there will be some
backfilling of the vasculature via the coronary sinus and Thebesian
veins but only a small amount of fixative will reach the heart muscle
via those routes.
A more direct route would be to enter the left ventricle with a
needle and then cut the right atrium or inf. vena cava. Ten or fifteen
seconds of perfusion will be sufficient to wash enough blood out of the
coronary vessles to get a good perfusion, you can see the heart change
color very quickly during this process.
However, I don't think perfusion mechanics are your problem. Try
fixing longer and at room temp and make sure your knife is very sharp.
Small pieces of heart should not need 5 days in sucrose to be
cryoprotected, 1-2 days should be plenty. Perhaps some gelatin in the
ventricular cavity would help support the endocardium?
Charles Scouten wrote:
> 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
>From: email@example.com [mailto:firstname.lastname@example.org] On Behalf Of Solverboy@aol.com
>Sent: Wednesday, April 28, 2004 10:52 AM
>Subject: [Histonet] Help With Cutting Heart
> 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,
>Dept. Neurbiology & Behavior
>SUNY Stony Brook
>Histonet mailing list
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Geoff McAuliffe, Ph.D.
Neuroscience and Cell Biology
Robert Wood Johnson Medical School
675 Hoes Lane, Piscataway, NJ 08854
voice: (732)-235-4583; fax: -4029
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