Abstract
SYNOPSISCatecholamines become intensely fluorescent when exposed to formaldehyde. This is due to closure of the side chain by a methylene bridge and subsequent dehydrogenation, which results in the formation of intensely fluorescent isoquinolines. Exposing freeze‐dried tissue to formaldehyde vapour makes it possible to study the distribution of catecholamines by fluorescence microscopy. The present paper deals with the practical performance of the formaldehyde‐induced fluorescence.The technique consists of the following steps: Freezing. This must be rapid. An apparatus is proposed for freezing at maximum rate, in which the tissue slice comes into sudden contact with a cold metal surface. Drying. The tissue must be dried at a temperature below −40°C, using an efficient apparatus. The types of apparatus are described and discussed. Small tissue pieces can be dried overnight with an efficient apparatus. Formaldehyde condensation. Formaldehyde is generated by warming paraformaldehyde powder in a glass vessel containing the freeze‐dried tissue pieces. The temperature of exposure and the water content of the paraformaldehyde powder must be properly adjusted for each tissue. Exposure for 1 hour at 50°C to paraformaldehyde powder equilibriated with 60 p.c. relative humidity is recommended as a start. Embedding, sectioning, and mounting. The formaldehyde‐treated tissue pieces are embedded in paraffin wax or, for better resolution, in epoxy resin. Sections must be spread on the slide without using water. Xylene, paraffin oil, and Entellan are suitable mounting media. Fluorescence microscopy. Any microscope fitted with non‐fluorescent optics is suitable for fluorescence work. A sufficiently intense light source is necessary; the high pressure mercury vapour lamp HBO 200 (Osram) is suitable. Special effects can be obtained with different filter combinations. The Schott filters BG 12 and OG 1 are suitable for most practical studies of catecholamines.
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