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CERTAIN TECHNICAL ASPECTS OF FLUORESCENCE MICROSCOPY AND THE COONS FLUORESCENT ANTIBODY TECHNIQUE

JOHN E. TOBIE 1

1 Laboratory of Immunology, N.I.A.I.D., National Institutes of Health, Bethesda 14, Maryland

A fluorescence microscope possessing an efficient ultraviolet light source, the 1000 watt A-H6, is described with special reference to its application in studies concerned with the fluorescent antibody technique and the tissue localization of the tetracyclines. The spectral characteristics of the light source are discussed and methods outlined whereby combinations of certain ultraviolet transmitting and absorbing filters can be used for maximum stimulation and visualization of fluorescent substances in tissue specimens. In fluorescent antibody studies when fluorescein isocyanate is used as the fluor, superior results have been obtained by excitation of tissue sections with blue-violet radiation at a wavelength band of 390-440 mµ rather than with ultraviolet radiation at 360-370 mµ. The two outstanding advantages of the longer wavelength stimulation are: 1) The increased fluorescence and consequent visual enhancement; and 2) the greater cellular detail for visual orientation and photographic clarity thus obtained. In contrast to this, the tetracyclines were more effectively stimulated in sections of bone with ultraviolet radiation at wavelengths in the 360-370 mµ band.

A method of quick-freezing fresh tissues in petroleum ether at approximately –65° C has been found more satisfactory for preserving the cellular architecture of tissues used in the fluorescent antibody technique. The operation of a rotary microtome in a cryostat at sub-freezing temperatures has been improved by special lubrication with the result that sections can be cut at a microtome setting of 3 µ. With these thin sections, it is possible to observe more minute cytological details and to localize fluorescent substances more accurately.

Submitted on January 28, 1958


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S. F. Kuvin, J. E. Tobie, C. B. Evans, G. R. Coatney, and P. G. Contacos
Antibody Production in Human Malaria as Determined by the Fluorescent Antibody Technique
Science, March 30, 1962; 135(3509): 1130 - 1131.
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