New tissue fixation method for cytochemistry using migrowave irradiation. I. General remarks.

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Abstract
We examined a rapid tissue fixation method using microwave irradiation (MWI) for ultrastructural, and cytochemical studies, including those on enzymeor immunolectin-cytochemistry, autoradiography, and X-ray microanalysis. MWI was applied to 0.3 to 1cm3 blocks of several kinds of rat tissues at 2.45GHz/500W for about 20sec in a fixative, as shown in the text, at room temperature. We tried to find why MWI enabled the aldehyde to penetrate into the tissue block so quickly. Microautoradiography was performed using 3H-formalin as a tracer. With MWI, prominently developed silver grains appeared homogeneously in the section, while the control section showed no silver grains within the section, only around it. Clearly, the microwave energy enables 3H-formalin to penetrate quickly into the tissue block. However, using biochemical analysis, we found that complete tissue fixation occurred only after the chemical crosslinking of proteins with aldehyde which was a gradual process. Thus, for good fixation by MWI, the tissue blocks needed to be left in the fixation medium for about 10 to 60min after MWI at room temperature for cytochemical fixation and for about 30 to 180min for conventional fixation. A fixative containing a small amount of tannic acid, 0.05 to 0.1%, is effective for fixing soluble peptides or proteins. A fixative containing NHA (see in the text), phosphate or oxalate can be used to detect the localization of Ca ion in tissue and cells. Combinations of these techniques have yielded excellent results in conventional light and electron microscopy, as well as in histochemical and cytochemical studies. MWI also yields very good results in high resolution electron microscopy, for example, many cholinergic synaptic vesicles at the presynaptic membrane in the rat brain could be observed for their opening images. The fixation effect of MWI seems to be equivalent to that of the rapid freezing fixation method. The enzymatic activity of alkaline phosphatase could withstand MWI treatment, even when the sample had been embedded in Technovit resin. However, acid phosphatase and glucose-6-phosphatase seemed to be rather weak against the MWI. Lectin binding sites also were stable against MWI treatment. MWI is excellent for micro- and electron autoradiography and also for X-ray microanalysis.