An improved method for studying microvascular geometry using fluorescent dyes: preventing dye extravasation, preserving dye integrity and enhancing tissue morphometry.
A procedure for stabilizing fluorescent markers used to study the microvascular geometry and morphometry of muscle tissue is described. The procedure involves fluorescent labeling of plasma, fixation of muscle tissue in 10% buffered formalin, and quick freezing. This procedure prevents extravasation of the fluorescent dyes out of the capillaries as frequently seen in other muscle microvascular techniques, thereby greatly increasing the time that capillaries are visible. We found that formalin may actually increase the rate of fluorochrome bleaching by photo-oxidation, but the increased rate of bleaching is more than offset by the greater concentration of dye trapped in the capillaries. Further, formalin fixation results in little distortion of the muscle fibers themselves, making this approach ideal for morphometric studies.
Department of Environmental, Population and Organismic Biology, University of Colorado, Boulder 80309-0334.
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CA
Farrelly
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Snyder
D000818:Animals; D002196:Capillaries / Q000648:ultrastructure; D015925:Cryopreservation; D005119:Extravasation of Diagnostic and Therapeutic Materials; D005404:Fixatives; D005456:Fluorescent Dyes; D016591:Histocytological Preparation Techniques; D008297:Male; D008856:Microscopy, Fluorescence; D009132:Muscles / Q000098:blood supply*; D016612:Paraffin Embedding; D051381:Rats; D017207:Rats, Sprague-Dawley
D005404:Fixatives; D005456:Fluorescent Dyes