Ectopic and heterotopic bone formation rarely occur in stromal tissues of various epithelial tumors. In the present study, ectopic bone forming tumors; calcifying epithelioma Malherbe (6), renal cell carcinoma (1), thyroid adenocarcinoma (1) and ovary carcinoma (1) were examined. The process of ectopic bone formation could be classified into 1) bone formation related to epithelial tissue as found in calcifying epithelioma of Malherbe, 2) bone formation from perivascular smooth muscle, and 3) that due to metaplasia occurring in stromal connective tissue of epithelial tumors. Immunohistochemical detection of proteoglycans (PG) was made in bone-forming matrix areas, and these areas were positive for chondrotin 4 and 6 sulfate, and dermatan and heparan sulfate PGs. Immunohistochemically, fibronectin, collagen III and bone sialoprotein as matrix proteins, and carbonic anhydrase as calcification marker were tested and they appeared in these pre-calcifying tissues.

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A panel of four mouse monoclonal antibodies, detecting blood group antigens of Lewis systems, has been used to define the immunohistochemical distribution of antigenic structures within normal, basal cell hyperplastic, and squamous metaplastic human bronchial epithelia. The reagents employed detect the following blood group specificities: Lewis a (Le^a), Lewis b (Le^b), Lewis x (Le^x), and Lewis y (Le^y).
In the normal bronchial epithelium, Le^a and Le^b were absent in all cases irrespective of Lewis phenotype. Le^y was present in all Le^a-b+ individuals and absent in the Le^a+b- and Le^a-b-. Le^x expression was not influenced by Lewis phenotype. In the squamous metaplastic epithelium, Le^b appeared in all cases irrespective of Lewis phenotype.
These immunohistochemical findings suggest that the absence of Le^a and Le^b is characteristic of the normal bronchial epithelum irrespective of secretor status of the patient. Le^y is also a marker for normal bronchial in the Le^a-b+ group, and Le^b is a marker for the squamous metaplasic epithelium.

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Previously, we found abundant filaments consisting of actin and cytokeratin in nonciliated bronchiolar epithelial (Clara) cells by treating the cells with detergent-containing medium. In this study, the presence of the filaments and the expression of actin mRNA in Clara cells of perinatal rats were investigated by electron microscopy and in situ hybridization, respectively. Clara cells in prenatal rat were characterized by the presence of glycogen and the absence of electron-dense granules. Detergent treatment did not clarify the presence of filaments in prenatal cells. After birth, there was a rapid loss of glycogen and electron-dense secretory granules appeared in the cytoplasm of Clara cells. Filaments could be recognized by detergent treatment. By the 3rd postnatal day, some Clara cells showed features similar to the cells in adult rats. To investigate whether these filaments demonstrated by detergent treatment were synthesized during the postnatal period, the localization of actin mRNA was observed using an in situ hybridization technique. Signals showing the presence of actin mRNA were observed in many cells at every stage of development. In the prenatal period, the signals were distributed diffusely in the epithelial cells of the bronchioles, but in the postnatal period, they appeared in Clara cells selectively. These results show that the actin filaments observed in the apical caps of Clara cells are synthesized during the postnatal period when Clara cells are thought to become mature.

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Acid phosphatase (AcPase) and thiamine pyrophosphatase (TPPase) have been detected with cerium as a capture agent for the first time in ciliated protozoa. In the trophozoites of the parasitic ciliate Ichthyophthirius multifiliis, AcPase activity was previously reported in food vacuoles, primary lysosomes, golgian-like cisternae and in some alveolar sacs. These former results obtained by lead methods, have now been confirmed and improved by a cerium-based technique. TPPase activity was also observed in golgian-like cisternae and alveolar sacs. As the cell membrane of this ciliate proved to be highly impermeable to cerium ions, several hr of preincubation in buffer with cerium chloride and Triton X-100 were needed to detect phosphatases with cerium in intact ciliates. However, in sectioned ciliates the preincubation time could be greatly reduced. Since TPPase became inhibited after a long preincubation, it could only be detected with cerium in sectioned ciliates.

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The development of Wistar rat fundic glands was studied using Griffonia simplicifolia agglutinin-IB₄ (GSA-IB₄) at the light microscopic and electron microscopic levels.
In adult rats, GSA-IB₄ specifically labeled the intracellular canaliculi and the apical surface on the fundic gland parietal cells, whereas the other sites of these cells were very weakly or rarely labeled.
Ontogenic studies revealed that at 18 days of gestation, GSA-IB₄ positive reaction was found along the whole cell surface of the cells on the fundic gland and immature intracellular canaliculi of the primitive parietal cells. At 1 day after birth, the positive reaction was restricted to the parietal cells. GSA-IB₄ specifically labeled the intracellular canaliculi and the apical cell surface of the parietal cells, but the labeling of the basolateral cell membrane of parietal cells decreased. At the point of weaning, 3 weeks after birth, GSA-IB₄ strongly labeled the intracellular canaliculi whereas the basolateral membrane was weakly labeled. This staining pattern did not change basically until adult stage.
From these results, it is concluded that the functional maturation of the parietal cells may occur at weaning in spite of their early appearance of morphological characters.

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The immunohistochemical distribution of superoxide dismutase (SOD) was examined in the human endometrium throughout the menstrual cycle. Surface and glandular epithelia showed positive staining throughout the menstrual cycle except just prior to the menstruation. Staining activity was more intense in the cytoplasm than that in the nucleus. Specific immunostaining of SOD was demonstrated in sub- and supranuclear vacuoles of glandular epithelia, and intraglandular substances during the early and mid secretory phases; i.e., the preand peri-implantation periods. Meanwhile, stromal cells showed weaker staining activity than surface and glandular epithelia throughout the menstrual cycle until the 22th day. Once the predecidual change occurred in stromal cells on the 23th day, predecidual cells came to show intensive staining. However, specific staining was shown neither in predecidual cells nor in surface or glandular epithelia just prior to the menstruation. In addition, SOD activity was also shown in the decidual cells of 8 weeks gestation and in the reserve cells of the endocervix. Collectively, the present immunohistochemical recults suggest that SOD may play an important role not only in the protection of developing embryos from superoxide anion radicals but also in the local defense mechanism against tissue damages resulting from inflammation in the uterine cavity, and that the expression of SOD may be regulated by sex steroids, especially progestorone. Furthermore, SOD could be a useful indicator to clinically diagnose the degree of predecidualization and the luteal phase defect.

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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.

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Microwave irradiation (MWI) was evaluated as a means of fixation for the detection of enzymatic activities such as alkaline phosphatase, acid phosphatase, and glucose-6-phosphatase in the liver and the kidney.
MWI enabled the detection of intracellular alkaline phosphatase enzymatic activities in perinuclear cisternae and rough endoplasmic reticulum cisternae of the hepatocytes; this had not been possible with the conventionally fixed cells. With MWI, acid phosphatase tended to be diffusible from lysosomes. This suggested that microwave energy had some effect on the membrane.
We tested MWI fixation also for autoradiographic studies using ³H-thymidine and ¹²⁵I-peptide. Excellent ultrastructural preservation and clear detection of radiolabelled compounds showed the MWI technique to be excellent and reliable for autoradiographic studies.

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The temperature during microwave irradiation in the core of tissue blocks, either 1cm or 2cm cubes of rat kidney and liver, was measured by plunging a sensor into the tissue blocks. The temperature of the tissue blocks was much higher than the outer media, i.e., chemical fixatives. Thus, the temperature of the outer media did not reflect that in the tissue block to be fixed. The temperature of tissue reached 40°C within 5-10sec of irradiation. The highest temperature of tissuee block at 15-second irradiation, for example, was 92.5°C (1cm cube of kidney) and 98°C (2cm cube of liver), using 4% PFA with CaCl₂ as a fixative. Microwave fixation is a practical tool for the preparation of tissue spections for either histology or cytology. However, it must be required that the irradiation is controlled so as not to produce high temperature within specimens.

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The usefulness of the specimens prepared following fixation by microwave (MW) irradiation in immunohistochemical reactions was evaluated using various antibodies. The results showed that in the case of the content of secretory granules, the antigenicities were found to be preserved well along with improved structural preservation by MW irradiation, and specimens with excellent localization were obtained. The antigenicity of basement membrane components could always be detected when tissues were fixed by MW irradiation with water soluble carbodiimide (WSC) added as a fixative. For plasma membrane antigens and cytoskeleton antigens, essentially the same results were obtained by fixation with MW irradiation and conventional fixation by immersion. There thus appears the possibility that the use of new fixatives may render MW irradiation more useful for immunohistochemical reactions.

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In the present paper, we demonstrated the presence of various cytokines in activated mononuclear cells immunocytochemically by using a rapid microwave (MW) fixation technique. Cytospin preparations of human peripheral blood mononuclear cells activated by a biological response modifier, OK-432, in vitro, were quickly fixed by MW irradiation in a diluted aldehyde solution consisting of 0.05% glutaraldehyde, 2% formaldehyde, 0.025% calcium chloride and 0.1M sodium cacodylate in phosphate-buffered saline (PBS) (pH 7.4). In the stimulated cells, tumor necrosis factor-α (TNF), interleukin-1α (IL-1α), and interleukin-1β (IL-1β) were clearly demonstrated immunocytochemically by using the specific antibodies for each cytokine.
Immunoreactivity for TNF appeared in the cells after 1.5hr of stimulation, and diffuse cytoplasmic stain was achieved after 24hr of stimulation.
IL-1α appeared in both cytoplasms and nuclei of the stimulated cells after 3hr. The distribution of cytoplasmic IL-1α suggested the association with the cytoplasmic membrane. With IL-β, a strong diffuse stain was obtained after 6hr, and indicated that the IL-1β was concentrated in cytoplasm.
Rapid MW fixation makes it possible to fix metabolically labile cytokines which have been difficult to localize in routinely fixed specimens. The method is expected to be a valuable tool in the study of cell biology.

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Application of microwave irradiation has been explored both in fresh and fixed frozen sections and in metallic stainings. The results showed a combination of irradiation alone followed by further irradiation immersed in any reagent containing about 50% alcohol made better microscopic-image quality in fresh frozen sections, but lesions of lymphoid tissues and lipid-rich brain tissues required further technical consideration for making better nuclear details and reducing spongy appearance. Fixed frozen sections had disappointing results with insufficient fixation and various degrees of freezing and mechanical damage. Standardization of fixed frozen sections may be a future problem. Metallic staining was time-saving and produced exellent results. A microwave oven can be a useful tool in surgical pathology.

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Microwave (MW)-stimulated fixation was applied to surgical pathology and preembedding immunoelectron microscopy. Fresh specimens were microwavedin the fixatives for 15-30sec using a conventional MW oven (500W) containing 200ml of water load. The temperature of fixatives was raised to 35-45°C. After MW radiation, the specimens were kept in the same fixatives for 1-4hr at 4°C or at room temperature as the postfixation procedure, which was important for the completion of chemical fixation of whole specimens. For light microscopical examination, buffered 10% formalin was used. Hematoxylin-eosin staining from paraffin-embedded specimens was completed within 24hr with excellent preservation of morphology and immunoreactivity. For conventional electron microscopy, MW fixation in 2.5% glutaraldehyde-2% paraformaldehyde showed excellent preservation of cell organelles. For immunoelectron microscopy by the preembedding method, 4% paraformaldehyde solution containing 0.1% glutaraldehyde was used, followed by one hr postfixation. Simultaneous improvement of both ultrastructure and antigenicity were obtained without any significant changes of the results. Our results indicate that MW-stimulated fixation with postfixation procedure is a useful tool for various fields of morphological investigations, and its beneficial effects were most remarkable for the preembedding immunoelectron microscopy technique.

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