[Histonet] mapping capillary perfusion pattern
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.
Biochemistry, medicine school
University of Tasmania, Australia
61 03 6226 2669
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