To analyze specific mRNA at individual cell level, non-radioactive in situ hybridization has been a powerful technique. In situ hybridization is most commonly done using double-stranded cDNA probes. We have already reported the usefulness of thymine-thymine (T-T) dimerized double-stranded cDNA as a non-radioactive probe. In this study, we extended this approach to use synthetic oligodeoxynucleotide (oligo-DNA). Two oligo-DNAs (I; 65-mer, II; 57-mer) complementary to the different regions of the human c-myc mRNA were synthesized and ultraviolet (UV)-irradiated (7, 000J/m²). When the T-T dimerized oligo-DNAs were dot-blotted to nitrocellulose filters, at least 40pg of both oligo-DNAs was detected immunohistochemically using anti-T-T antibody. By dot-blot hybridization, about 80pg of sense strand of c-myc DNA was specifically detected with T-T dimerized c-myc oligo-DNA(I). When the oligo-DNA (II) was used as a probe, however, the sensitivity of signal detection was significantly lower. Furthermore, the c-myc antisense oligo-DNAs reacted with the total RNA from HL-60 cells, but not from normal rat liver. Finally, using these oligo-DNAs, c-myc mRNA was detected in HL-60 cells.

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In situ hybridization technique was applied to identification of the cells producing structurally related hormones, gastrin and cholecystokinin (CCK). By this technique using biotinylated DNA probes and alkaline phosphatase histochemistry, we looked for the localization of each messenger RNA (mRNA) for the hormones on tissue sections of biopsy specimens of human gastric antrum and duodenum. As a result, gastrin mRNA positive cells were found in the pyloric glands, while CCK cells were not detectable in the gastric antral mucosa. In the proximal duodenum, CCK- and gastrin-cells were detected separately. In conclusion, detection of mRNA by in situ hybridization was considered to be a useful means for analyzing endocrine cells in the gut.

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Tissue distribution and metabolic fate of ¹⁴C from [1-¹⁴C]cystine and ³⁵S from [³⁵S] cysteine, both precursors of taurine, in various tissues and organs of pregnant mice were studied by whole-body autoradiographic and biochemical analysis.
Autoradiography of whole-body cryosection was performed at 30min, 3 and 6hr after i. v. injection of radioactive sulfur amino acids ([1-¹⁴C] cystine and [³⁵S] cysteine). In the maternal body, the highest optical density (OD) was observed in the pancreas and then the kidney, small and large intestines, stomach, Harderian gland, liver, salivary gland and mammary gland followed.
Low ODs were observed in the brain and skeletal muscle. ODs of ¹⁴C and ³⁵S in the placenta were relatively high. In the fetus, high ODs of ¹⁴C were observed in the lens and ³⁵S in the cartilage and lens.
In the biochemical analysis, the mice were decapitated at the same intervals after injection as those for autoradiography and various tissues were removed. These tissues were fractionated into 6% perchloric acid-soluble, -insoluble and lipid fractions. Total radioactivities (cpm/g wet weight) of ¹⁴C and ³⁵S were high in the kidney, pancreas, Harderian gland and stomach, and low in the brain, skeletal muscle and fetus. The liver and bile showed high radioactivities in ³⁵S, but low radioactivities in ¹⁴C. These data were in good agreement with those of whole-body autoradiographs. The radioactivities incorporated into the acid-in-soluble fraction were high in the pancreas, stomach, large intestine and salivary gland, but low in the liver, kidney and skeletal muscle after injection of [1-¹⁴C] cystine and [³⁵S] cysteine. The acid-soluble fractions of the liver, kidney, pancreas and small intestine were analyzed by thin-layer chromatography. The radioactive spots for cysteine, cysteine sulfinic acid (CSA) and taurine were detected, but the radioactive level and its change with time in each radioactive spot were different among the organs.

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Developmental changes in the distribution of parvalbumin immunoreactivity in the rat reticular thalamic nucleus from embryonic day 18 to adult were studied using immunohistochemistry. Neurons containing parvalbumin immunoreactivity in their cell somata were first detected at embryonic day 18. By embryonic day 20, many cells extended parvalbumin-positive fibers into the ventral thalamic nucleus. During development of the ventral thalamic nucleus, these fibers increased in number and thickness and ran in various directions. By postnatal day 16, these fibers became thinner and less well-stained. Neurons in the reticular thalamic nucleus extended their parvalbumin-positive axons into other nuclei of the thalamus, but in these other nuclei, parvalbumin-positive terminal and fibers developed primarily postnatally. In the reticular thalamic nucleus, the distribution of GABA-like immunoreactivity was very similar to that of parvalbumin at embryonic day 20 with cells extending fibers which contained immunoreactivity for GABA into the ventral thalamic nucleus. Thus parvalbumin immunoreactivity appears in the neurons of the reticular thalamic nucleus early in development of this nucleus, first in cell somata of neurons, then in their axons and dendrites. Apart from the ventral thalamic nucleus, the innervation by parvalbumin containing neurons in the reticular thalamic nucleus of the rest of the thalamus, showed a variable developmental pattern with different nuclei being innervated at different times, presumably reflecting the separate development of individual thalamic nuclei.

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Intermediate filament-rich cytoskeletal protein was extracted from normal and 7 or 21-day bile duct-ligated rat hepatocytes, and analyzed by both one and two-dimensional gel electrophoresis. The most marked change in the bile duct-ligated rats was an increase in 44kd polypeptide, determined by immunoblotting to be actin. Cytokeratin, the intermediate filament type found in hepatocytes, was also slightly increased. However, a marked decrease of the basic side spot of 55kd paired cytokeratin components was noted on two-dimensional gel electrophoresis. Fluorescent staining of liver tissue F-actin using NBD-phallacidin showed no remarkable difference between the two groups. Therefore, the increased actin content of hepatocyte after bile duct ligation may be mainly G-actin rather than F-actin. These changes in hepatocyte cytoskeleton seem to be a cellular adaptation to the microenvironment.

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Immunolocalization of both copper-zinc (CuZn) and manganese (Mn) superoxide dismutases (SODs) in various formalin-fixed rat tissues was investigated using the indirect immunoenzyme method. The optimal dilution was 1:10000 for rabbit anti-rat CuZnSOD and 1:5000 for anti-rat MnSOD antisera, respectively. The higher dilutions of the present antisera than those used in previous studies enabled a higher resolution of the present immunostaining because of a lower background staining. Neither SOD have been previously reported to be stained in eyeballs and brain. The following tissues were intensely stained for CuZnSOD: the fundic glands of the stomach, epithelium of the large intestine, tracheal and bronchial epithelia, liver parenchymal cells, proximal tubules and papillary ducts of the kidneys, pancreatic ducts and islets, zona glomerulosa of the adrenals, myocardial cells, ciliary bodies and pigment cell layers of retinas. On the other hand, the MnSOD reactivity was found to be rich in the following tissues: the gastric pits, epithelium of the small intestine, tracheal epithelium, Henle's loop and collecting ducts of the kidneys, zona fasciculata of the adrenals, ciliary bodies and inner plexiform layer of retinas, and cortical neurons. The surface epithelia of both gastrointestinal and respiratory tracts tended to have high reactivities for both SODs, suggesting that the antioxidant enzyme defense system in these tissues responded to the exposure to superoxide generated from environmental oxygen. There was a wide range of variability in the distribution of each SOD from cell to cell. The present technique is thus considered to be useful for revealing site-specific changes in SODs in various rat tissues.

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The localization of gamma-glutamyl transpeptidase (γ-GTP) in the rat kidney was examined by immunoelectron microscopy using monoclonal antibody to rat γ-GTP. The enzyme was distributed along the brush border and in the apical canaliculi and vacuoles of the proximal tubules. The immunoreaction of γ-GTP in the brush borders increased from the S₁ to S₃ segments in the proximal tubules. Conspicuous γ-GTP immunostaining was observed along the basolateral membranes of the S₂ and S₃ segments of the proximal tubules. In addition, the apical cell membrane of the upper part of descending thin limbs of Henle's loops exhibited positive γ-GTP immunostaining. A faint reaction was occasionally recognized in the apical cell membranes of the distal convoluted cells, including the macula densa.

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Monoclonal antibody for keratin K8.12 (keratin nos. 13 and 16) was evaluated in paraffin sections of 17 cases of obstructive sialadenitis and 97 pleomorphic adenomas. In normal salivary glands fixed with 10% formalin, K8.12 staining was strongly limited to ductal basal cells, and staining to PKK1 and KL1 was positive in duct cells. Monoclonal antibodies PKK1 and KL1 were compared in duct-like structures of obstructive lesions and pleomorphic adenomas. Duct-like structures in obstructive lesions showed strong staining for K8.12 in ductal basal cells, and positive staining for PKK1 and KL1 in ductal epithelial cells, in different stages of ductal obstruction. Pleomorphic adenomas showed an intense expression for K8.12 staining in outer tumor cells of tubulo-ductal structures, and positive reaction for PKK1 and KL1 in luminal tumor cells. The histogenesis of pleomorphic adenoma is discussed in terms of the immunohistochemical labelling of the 3 types of monoclonal antibodies to keratin. The identification of a progenitor cell to ductal basal cells is also hypothesized.

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The electron microscopic localization of the AChE-enzyme activity in some endocrine cells of the rat pancreas was studied using the method of Lewis and Shute (1966) and the diisopropyl fluorophosphate (DFP) pharmacohistochemical method. The results obtained show that some A-, D-, PP- and B-cells situated at the periphery of the islets of Langerhans contain AChE activity. The electron opaque material was distributed within the cisterns of the rough endoplasmic reticulum and the nuclear envelope. Different parts of the halo of single secretory granules were also filled with reaction precipitate. In the animals treated with DFP no enzyme activity was observed during the first 4hr. Six hr after the injection of DFP the enzyme activity recovered and the deposition of the reaction product was again visible in the usual locations. These results show that some endocrine cells of the rat pancreas synthesize AChE.
It is suggested that the AChE of the pancreatic endocrine cells most probably plays a non-cholinergic role. The importance of the enzyme for the processing of some endogenous or exogenous hormones and regulatory peptides is emphasized.

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Ultrastructural immunolocalization of human thrombomodulin was visualized for the first time in the human placenta; i.e. plasma membrane of microvilli of syncytiotrophoblast was continuously positive with occasional reactivity along the membrane of intracytoplasmic vacuoles. This is consistent with its physiological property as a receptor to thrombin.

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The localization of aldehyde dehydrogenase (ALDH) activity in the hapatic lobule of normal mice was studied histochemically by the copper ferrocyanide method.
Reaction products, brown copper ferrocyanide showing the enzymatic activity, were mainly found in the centrilobular zone. The reaction was significantly inhibited by disulfiram (100μM), but was never fully eliminated.
This study suggests that aldehyde dehydrogenase activity, as well as alcohol dehydrogenase activity is localized mainly in the perivenous zone of the hepatic lobule in normal mice.

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The double staining procedure using both the nitroblue tetrazolium (NBT) and copper ferrocyanide methods was presented for the simultaneous localization of two different dehydrogenase activities in a single histologic section.
Sections of normal mouse liver were stained for alcohol dehydrogenase (ADH) activity using the NBT method first, then stained for aldehyde dehydrogenase (ALDH) activity using the copper ferrocyanide method. However, the formazan showing ADH activity must chelate metal ion (Cu²⁺, Co²⁺, or Ni²⁺) prior to staining for ALDH activity.
Both the blue metal-formazan complex showing ADH and brown copper ferrocyanide showing ALDH activities in a single liver section were localized mainly in the perivenous zone of the hepatic lobule. In addition, ALDH activity is always localized in the zone of the lobule where ADH activity is present.

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