[Histonet] mapping capillary perfusion pattern


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

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