As an extension of our ultracytochemical studies on the cell membrane, phosphotungstic acid (PTA) staining dissolved in various media was applied to the microvillus membranes of the cat small intestine. The staining was performed on ultrathin epon sections of fixed specimens. In many cases oxidation or bleaching of epon sections by performic acid (PF), hydrogen peroxide (PO) or periodic acid (PA) was done prior to the PTA staining.
In general, the PFP (PTA dissolved in ddw or HCl) method, the POP (PTA dissolved in ddw or HCl) and the PAP (PTA dissolved in chromic acid) gave good stainability of epon sections. However, among these three methods the most reproducible results were obtained by the PFP method.
The PFP reaction was positive in the glycocalyx, the apical and lateral membranes, apical vesicles, the maturing face of the Golgi complex and lysosomes of the absorptive epithelia and the goblet cells, mucigen granules in the goblet cells, granules of basal granulated cells and capillary endothelia, reticular and collagen fibrils and the basement lamina. It was of great interest to find an electron density spanning the plasma membrane in addition to the unit membrane-like structure.
In order to elucidate the chemical nature of the PFP positive substances spanning the plasma membrane the PFP reaction was performed on specimens fixed in various fixatives, digested by various enzymes or extracted in an organic solvent, and on pure model samples. The data obtained thus far seem to indicate that the electron density spanning the plasma membrane observed in specimens stained by the PFP method is due to phospholipids and/or glycolipids in the membrane.
It may also be stressed that the PAP, POP, or PFP reactions in general, and in particular the PFP reaction because of its high reproducibility, are extremely useful as conventional substitutes for the periodic acid-Schiff (PAS) -type reaction under the electron microscope, and can be performed quite easily on epon sections.

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The originating neurons of the ipsilateral efferent fiber connections from retrograde transport of horseradish peroxidase (HRP). The origin of the efferents from the amygdaloid complex to the preoptic area are the neurons in the central and medial nuclei. The cortical nucleus and other nuclei of the amygdaloid complex could not be ascertained as the origins of these efferent fibers in the present study.

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Two different isozymes of lactate dehydrogenase, skeletal muscle type (LDH-M₄) and cardiac muscle type (LDH-H₄), were purified to a high degree from rat skeletal muscle. The distribution of each isozyme in rat liver was investigated by use of the peroxidase-labeled antibody method at light and electron microscopic levels. Using histochemical technique, the final reaction deposits were recognized in the cytoplasm, near the cell membrane facing both Disse's space and bile canaliculi, and in the mitochondria. By using the peroxidase-labeled antibody method, LDH-M₄ was demonstrated in an amorphous part of the cytoplasm of the parenchymal cells, which might correspond to the glycogen area. On the other hand, LDH-H₄ isozyme was localized in amorphous cytoplasm near the cell membrane facing both the bile canaliculi and Disse's space, and in the mitochondria of parenchymal cells.
In both histochemical and immunohistochemical studies, lactate dehydrogenase was recognized neither in the stellate cells of Kupffer nor in the endothelial cells of the sinusoid.

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The localization of hyaluronidase in the sheep testis was investigated by use of the peroxidase-labeled antibody method on the light and electron microscopic levels. The antigen was recognized in the acrosome of both spermatid and spermatozoon. In the electron microscopic observation, the antigen in the spermatid was concentrated in the acrosomal cap, but was not recognized in the acrosomal collar. However, in the spermatozoon, the antigen was recognized homogeneously both in the acrosomal cap and in the acrosomal collar. The results of the present study suggest that hyaluronidase localized in the acrosomal cap spreads into the acrosomal collar region as spermatogenesis proceeds from spermatid to spermatozoon.

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The localization of acrosomal proteinase in sheep testis was investigated by staining the tissue with peroxidase-labeled trypsin inhibitor on the light and electron microscopic levels. In spermatid and spermatozoon, the specific reaction was recognized in the acrosomal cap, although not in the acrosomal collar. These results indicate that the localization of acrosomal proteinase is different from that of hyaluronidase.

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The purpose of this investigation is to clarify the present situation of histochemistry and cytochemistry in medical and dental education, which has never been studied before, in order to present suggestions for improvement.

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