RE: [Histonet] better perfusion and inclusion---thanks to y'all
We have recently become aware that a critical error was made in the first manual of the Perfuison One. Sucrose in water is isotonic with body fluids at 9.25% sucrose by weight in water, or 92.5 grams of sucrose in distilled water. The manual incorrectly stated 5% sucrose to be isotonic. Proportions at which various solutions are isotonic with body fluids can be found at the following website:
Using a hypotonic prewash solution (5% sucrose in water is hypotonic) will cause water to enter the cells, which will cause them to swell, and to be cross linked to neighboring cells when the fixative arrives. The end result will be shrinkage when the tissue is stored in an isotonic solution later, by a different mechanism than isotonic sodium in the prewash and extracellualar fluid causes shrinkage, but the same end result. A slightly hypertonic solution, 10%, would be good protection for cells. They would shrink away from each other slightly, then return to normal size after fixation.
We apologize to any of you who have experienced this problem. The manual and the application note which are posted on the website have both been corrected.
The error arose because Cragg used 5% sucrose in PBS buffer, to make up an isotonic solution. Any two isotonic solutions may be mixed in any proportion, and if nothing precipitates out, the result is isotonic. However, PBS buffer contains some sodium. The purpose of the sucrose prewash is to reduce extracellular sodium to at or below intracellular sodium concentrations.
My use of the Perfusion One type apparatus, which avoided shrinkage and yielded excellent tissue quality and HRP reactions, was done several years ago with 10% sucrose in water. Recent testing which resulted in the picture on our website was done with 5% sucrose in buffer made up as an isotonic solution.
Extracellular fluid has about 140 mM sodium ion concentration. Intracellular sodium is hard to measure, but about 5-20 mM, or about 10% of the sodium in extracellular fluid, due to the action of the sodium pump proteins in the membrane. Sodium concentration in the prewash solution should theoretically be at or below the intracellular concentration to avoid cellular swelling Do not use any buffering which would result in sodium concentrations above 10 mM.
For anatomy work, HRP reactions, lesion location, and most applications, 10% sucrose in distilled water is the preferred solution. However, many antibody reactions run best in high salt concentrations, and at a preferred pH.
Question: Would a few seconds exposure to unbuffered sucrose solution, followed by fixation, followed by a return to buffered solution and proper pH, damage the reactivity permanently, or is it only the salt concentration and pH present during the reaction that is important?
Note that sodium is a problem only at the start of fixation, when it inactivates the sodium pump before the membrane is fully permeable to all ions.
To my knowledge, this question is testable, but not yet tested. If you have an answer, I would appreciate hearing it.
Sodium concentration in the prewash solution must be at or below the intracellular concentration to avoid cellular swelling. Do not use any buffering which would result in sodium concentrations above 10 mM.
One way to achieve this would be to prepare isotonic sucrose and isotonic phosphate buffer at the proper pH, and mix them 900 ml sucrose solution, 100 ml phosphate buffer. This would give a pH adjusted buffered solution, at the highest sodium ion concentration consistent with avoiding cellular swelling.
Another strategy would be to prepare an isotonic buffered, pH adjusted solution using buffers that do not contain sodium. Tell us your recipes if this does (and if it doesn't) work for you and they will be added to the manual referencing your lab.
Charles W. Scouten, Ph.D.
5918 Evergreen Blvd.
St. Louis, MO 63134
Ph: 314 522 0300
FAX 314 522 0377
From: firstname.lastname@example.org [mailto:email@example.com] On Behalf Of Nancy.Walker@sanofi-synthelabo.com
Sent: Thursday, June 10, 2004 6:53 AM
Subject: [Histonet] better perfusion and inclusion---thanks to y'all
Just to say thanks to numerous contacts that made helpful suggestions and give feed back.
*Thanks Gail Callis for her suggestions to reduce inclusion schedules .
This did in fact reduce dilation of mouse brain ventricles.
*Thanks to helpful suggestions of Charles Scouten, Geoff McAuliffe and Christophe Guerin, we greatly improved our perfusion technique with mouse brain resulting in better histology : less blood vessel collapse, cytoplasm retraction visual particularly around oligodendrocytes but also neurons, and better integrety of hippocampal cell layers.
What worked for us?
We set up a perfusion system based on physiological blood pressure which will take into account size variation between young and older animals. It's simple and cheap, too! Just supply pressure with the rubber bulb and manometer ( we used a medical blood pressure unit, without the cuff!) remember to use stiff tubing and adjust the catheter to just fit into the aorta of the size of your animal. Don't worry you don't have to pump much to keep up the pressure, especially with mice (a little more with rats).
This system resulted in better evacuation of red blood vessels than perfusing at 2.5 ml/min (110mm Hg in our 35g mice resulted in a speed of about 1.3 ml/min).
Trials to break the blood-brain barrier with brief treatments of sacharose or mannitol with high pressure did nothing to improve the histology rather made it worse. What did give more reproducible results in terms of histology was extending the perfusion time from 10 to 20 minutes. I put up a picture in the Histonet web gallery (http://pathcuri1.swmed.edu).
So in conclusion systems like Perfusion One that allow you to perfuse on a pressure basis do better flush out red blood cells, breaking the brain blood barrier with various treatments didn't help but maybe we didn't do it right. Longer perfusion times really reduce variability.
B.P. 37 Labége Innopole
31676 LABEGE CEDEX FRANCE
tel : (33)561004179 fax :(33)561004001
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