[Histonet] perfusion question

[John Kiernan]

The wash-out solution should have pH and osmotic pressure close to those of the animal's extracellular fluid, to avoid shrinkage or swelling of cells, collagen fibres etc. This can be achieved with simple saline (0.9% NaCl). A buffer prevents acidification of the extracellular fluid by products released from dying cells. Calcium ions (not compatible with phosphate buffers) enhance the preservation of phospholipids of cell membranes, myelin etc. Potassium ions are included in "physiological" saline solutions such as Ringer-Locke in which tissues and small organs can be kept alive, sometimes for several hours. I don't know of any study of effects of potassium on fixation, but probably someone has looked into it.

The formaldehyde should also be dissolved in an isosmotic buffer because the chemical events of fixation occur slowly (several hours). Brain tissue still responds to changes in ambient osmotic pressure after several hours in neutral buffered formaldehyde. In glutaraldehyde, however, the cells are stabilized in 20 minutes. See: Paljarvi L, Garcia JH, Kalimo H (1979) The efficiency of aldehyde fixation for electron microscopy: stabilization of rat brain tissue to withstand osmotic stress. Histochem. J. 11: 267-276. This paper has also has references to several other studies. 

Traditional fixative mixtures are mostly acidic and rapidly acting, stabilizing the structure of the tissue (for light microscopy) before the development of adverse effects of low pH or osmotic pressure. The subject was also reviewed by J.R.Baker in his book Principles of Biological Microtechnique (1958), pp.75-86.


John Kiernan
Anatomy, UWO
= = =
----- Original Message -----
From: Neil Fournier <nfournier <@t> sasktel.net>
Date: Wednesday, December 10, 2008 14:42
Subject: [Histonet] perfusion question
To: histonet <@t> lists.utsouthwestern.edu

> Is there a rationale for using normal saline (0.9% (w/v) NaCl 
> dissolved in dH2O) over 0.1 M PBS (pH 7.4) as a rinsing solution 
> during intraventricular perfusion of a rat. Would one yield 
> better results over the other?
> 
> Also is there a raionale for why some people perfuse using PBS 
> made only from monobasic and dibasic sodium phosphate (with 0.9% 
> NaCl) vs. using PBS that also include KCl, sodium phosphate 
> dibasic, NaCl, and potassium phosphate monobasic in the recipe.
> 
> Thanks for the help
> 
> Neil 
> _______________________________________________
> Histonet mailing list
> Histonet <@t> lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet



----- Original Message -----
From: Neil Fournier <nfournier <@t> sasktel.net>
Date: Wednesday, December 10, 2008 14:42
Subject: [Histonet] perfusion question
To: histonet <@t> lists.utsouthwestern.edu

> Is there a rationale for using normal saline (0.9% (w/v) NaCl 
> dissolved in dH2O) over 0.1 M PBS (pH 7.4) as a rinsing solution 
> during intraventricular perfusion of a rat. Would one yield 
> better results over the other?
> 
> Also is there a raionale for why some people perfuse using PBS 
> made only from monobasic and dibasic sodium phosphate (with 0.9% 
> NaCl) vs. using PBS that also include KCl, sodium phosphate 
> dibasic, NaCl, and potassium phosphate monobasic in the recipe.
> 
> Thanks for the help
> 
> Neil 
> _______________________________________________
> Histonet mailing list
> Histonet <@t> lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet