Re: [Histonet] mapping capillary perfusion pattern

From:Geoff McAuliffe

Dear Lei:

    Capillary bed perfusion varies depending on many local (microscopic) 
factors. Even in a hindlimb at rest capillary perfusion will vary with 
time and perfusion will depend on factors you probably can't measure. I 
would suggest not perfusing with anything; clamp the major vessels to 
trap the blood in the capillary beds. Then either fix or freeze and use 
benzidine or diaminobenzidine to demonstrate the hemoglobin in the red 
cells trapped in the capillaries. Someone did  this many years ago, 
published their method in the journal Stain Technology (now known as 
Biotechnic and Histochemistry), I think they were looking at vasa recta 
in the kidney.


zhangl wrote:

> Hi Histonetters,
> I am currently doing a project to map the capillary perfusion pattern 
> in perfused rat hindlimb. In this system, aorta and vena carva are 
> cannulated and single hindlimb is perfused wtih BSA buffer. What I 
> have tried was to infuse GSL-1 ( a glycoprotein shown to specifically 
> bound to small vessels ) for 30min, thus those perfused capillaries 
> would be labeled with GSL-1. Then I cut frozen muscle sections and 
> stain them with GSL-1 antibobidies etc. What I found was nearly all 
> available capillaries were labeled. I suspect that since GSL-1 was 
> infused for so long time,  some capillaries having trivial flow thus 
> not being functionally perfused were labeled as well. This method 
> could give me overestimated number of perfused capillaries. Has anyone 
> have similar experience?
> Next idea is to perfuse 2.5% glutaraldehyde at constant pressure for 
> 5min. Then I stain frozen muscle sections with GSL-1. Those perfused 
> capillared will be fixed and keep open, unperfued capillaries would 
> remain closed and appear as a dark dot on the sections. During the 
> perfusion fixation, perfusion pressure goes up presumably due to the 
> cross-linkage in perfused vessels, I need to reduce the perfusion flow 
> rate accordingly to keep pressure constant, also not let red blood 
> cell be washed out because this means recruiting unperfused 
> capillaries, in turn altering flow pattern. Although glutaraldehyde is 
> perfused for a fixed period, the total amount glutaraldehyde going 
> through vascular bed is different for each experiment due to the 
> variation in perfusion flow. My question is in my case whether the 
> time of perfusion fixation or the amount of fixative mainly determines 
> the degree of fixation? Has anyone similar experience? any suggestion 
> and advice is greatly appreciated.
> Lei Zhang
> Biochemistry, medicine school
> University of Tasmania, Australia
> 61 03 6226 2669
> _______________________________________________
> Histonet mailing list

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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