Attempts to combine 2-deoxyglucose autoradiography and tyrosine hydroxylase immunohistochemistry.
The possibilities were analysed to combine the 2-deoxyglucose technique and indirect immunofluorescence histochemistry using tyrosine hydroxylase antiserum, with the aim to study functional activity in immunohistochemically characterized single neurons. Since the product measured with the 2-deoxyglucose method is water soluble and since immunohistochemistry requires that sections repeatedly run through aqueous media, the 2-deoxyglucose method was carried out before fixation and immunohistochemistry. The routine rapid thaw-mounting at + 60 degrees C of sections for 2-deoxyglucose autoradiography was found not to be compatible with immunohistochemistry. Instead a new mounting technique based on "gluing" the sections on to the object slide with a mixture of a standard mounting medium (Permount) and xylene was used to avoid diffusion at this stage. Two procedures were outlined, both starting with unfixed brains cut on a cryostat. In Method I autoradiographic sheet film was used. After autoradiographic exposure, the section was immersion-fixed in formalin, processed for immunohistochemistry, analysed and photographed in a fluorescence microscope and the results compared with the autoradiographic distribution patterns on the film. However, only the low resolution of the routine 2-deoxyglucose technique was obtained, which did not allow analysis of activity in single cells. In Method II, liquid emulsion applied by the loop technique was used. After exposure, autoradiographic developing and fixation, dehydration, mounting, analysis and photography of autoradiographs in the light microscope, the cover-slip was removed, the sections rehydrated and processed for indirect immunofluorescence histochemistry. With this procedure single autoradiographically labeled cells were observed, some of which contained tyrosine hydroxylase. Thus, with Method II it may in the future be possible to monitor functional activity in single immunohistochemically identified neuronal cell bodies. In order to obtain a useful and reliable method for this purpose, however, further extensive work with regard to, for example, quantification will be required.
D000818:Animals; D001345:Autoradiography / Q000379:methods; D001921:Brain / Q000201:enzymology / Q000378:metabolism*; D003837:Deoxy Sugars / Q000378:metabolism*; D003847:Deoxyglucose / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006651:Histocytochemistry / Q000379:methods*; D008297:Male; D051381:Rats; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism*
D003837:Deoxy Sugars; D003847:Deoxyglucose; D014446:Tyrosine 3-Monooxygenase