A simple and useful double staining method has been developed for light microscopic immunohistochemistry. The technique consisted of a sequence of protein A-gold staining followed by a single silver enhancement procedure, and in this technique reaction products were colored black, and brown or reddish purple, respectively. The results obtained in the rat pancreatic islets and adenohypophysis in combination with a variety of histochemical controls have substantiated the specificity, reliability and reproducibility of the double staining method. The present method is simple and economical, since visualization of the reaction products of two different shades can be obtained by a single procedure.

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Metallothionein (MT), a low molecular weight intracellular protein present in cytoplasm and nucleus, possesses a selective binding affinity for zinc, copper and other group II heavy metal ions which may have a potential role to bind, sequester or detoxify these metal ions and alter the treatment response to chemotherapy or radiotherapy. The present study describes expression of MT in normal salivary glands and neoplastic salivary lesions using immunohistochemical method. Immunoreactive MT in normal salivary glands was observed in ductal cells where the immunostaining was most prominent in ductal basal cells of excretory and striated ducts and intercalated duct cells. Expression of MT was also observed in various salivary tumors; pleomorphic adenoma had immunoreactive MT in fibrillar and plasmacytoid neoplastic myoepithelial cells and chondroid cells whereas Warthin's tumor, monomorphic adenoma and adenoid cystic carcinoma had non-luminal or basally located tumor cells intensely reactive. Acinic cell carcinoma cells were weakly reactive, however, papillary cystadenocarcinoma had a mixed population of cells with majority of the tumor cells moderately, and few cells strongly reactive. The profile of distribution of MT in salivary gland tumors was similar to that for S100 proteins suggesting their coexpression. The result of the present study suggests that MT is expressed in normal salivary glands. In various salivary gland tumors, in spite of histopathological heterogeneity, differentiation, binding potential and sequestration or detoxification of toxic metal ions by MT may confer a heterogeneous biological behavior. They also respond to the therapeutic modality of radiation and chemotherapy.

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Objectives: In adenomotous hyperplasia (AH) and oncocytic adenomatous hyperplasia (OAH) of the intranodal heterotopic salivary gland tissue and Warthin's tumours of the parotid glands, the expression of various intermediate filament proteins and other tissue markers in the epithelial components were compared, since they share common histopathological characteristics with both eosinophilic luminal cells and basal cells.
Materials and Methods: Tissue specimens from 79 intraparotid and extraparotid cervical lymph nodes containing heterotopic salivary gland tissue and 10 Warthin's tumours of parotid glands were evaluated for the expression of cytokeratins, vimentin, actin, epithelial membrane antigen (EMA), S-100 protein, lysozyme (Ly), lactoferrin (La), α1 -antichymotrypsin (α1-ACT), α1 -antitrypsin (α1 -AT), and human gastric and lung cancer antigens by using the three stage streptavidin-biotin immunoperoxidase method.
Results and Conclusions: A similar profile of positive immunoreactivity for cytokeratins, EMA, Ly, La, α1 -ACT, α1 -AT and human lung and gastric cancer antigens was observed in the epithelial components of AH, OAH and Warthin's tumours, while vimentin, actin and S-100 protein were negative in all of them. The histopathological and immunohistochemical profiles of AH and OAH in the intranodal heterotopic salivary gland tissue were closely mimicked by the epithelial tumour cells in Warthin's tumours. These findings suggest that Warthin's tumours may sometimes, but not always, arise from the ductal cells of heterotopic salivary gland tissue included in the lymph nodes.

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Localization of steroid 5α-reductase type 2 in human prostate, seminal vesicle and vas deferens was investigated by light and electron microscopic immunohistochemistry using a rabbit polyclonal antiserum raised against a synthetic peptide fragment representing amino acids 225-240 of the human 5α-reductase type 2 enzyme. Under light microscope, the enzyme was localized in the cytoplasm of basal epithelial and stromal cells of the prostate. Secretory epithelial cells were negative. There was no immunoreaction in cell nuclei. Double immunostaining for 5α-reductase and muscle specific actin indicated that all smooth muscle cells had 5α-reductase type 2. However, a few type 2-positive cells did not react with a muscle specific actin antibody. Electron microscopic immunocytochemistry revealed that the cytoplasm of smooth muscle cells and of a few fibroblasts had immunoreaction products for this enzyme. In the seminal vesicle and vas deferens, basal and smooth muscle cells were positive for the type 2 enzyme. These findings indicate that the major sites of the DHT production are basal epithelial cells and stromal smooth muscle cells in human prostate, seminal vesicle and vas deferens. These cells may play a role in maintaining the functional activity of these male accessory sex organs through the production of DHT.

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The localization of the low-molecular-weight heavy-metal-binding protein metallothionein (MT) in the thyroid gland and medullary thyroid carcinoma was investigated by an immunohistochemical technique, and the amount of MT in the thyroid gland was assayed by radioimmunoassay.
MT was immunohistochemically identified in epithelial cells of follicular and parafollicular cells. Both the cytoplasm and the nuclei of the cells stained positive for MT. The nuclear staining for MT was strong. The follicular epithelial cells and colloid were positive for thyroglobulin, and the parafollicular cells were strongly positive for calcitonin.
Medullary thyroid carcinoma was strongly positive for calcitonin, but most tumor cells were negative for MT. MT-positive cells in the tumor were sporadic, and both their nucleus and cytoplasm stained positive.
Radioimmunoassay revealed high levels of MT in the thyroid gland, about 148.7±45.0 μg/g wet weight.

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The effect of colchicine on the intracellular transport of alkaline phosphatase (ALP) in primary cultures of adult rat hepatocytes was examined by immunocytochemical methods. In hepatocytes cultured in control medium, the reaction products indicating the presence of ALP were observed in the plasma membrane surrounding intercellular spaces, although they were also observed in the cytoplasm near the nuclei and in the plasma membrane along cell borders between adjacent hepatocytes. When hepatocytes were cultured in the above medium to which colchicine had been added to a final concentration of 10^-5M, the reaction products were mainly observed in several large autolysosome-like granules in the cytoplasm. Furthermore, upon examination by a fluorescence-labeled antibody method, microtubules were observed to extend in a radial pattern from the nuclei to the cytoplasm in hepatocytes cultured in control medium but they were not observed in hepatocytes cultured in the colchicine-supplemented medium. In the present study, based on morphological evidence, it was confirmed that the transport of ALP from the cytoplasm to the plasma membrane of adult rat hepatocytes is inhibited by colchicine.

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We have studied the keratin expression in normal and abnormal uterine cervix using the immunohistochemical method. Six commercially available keratin antibodies (M 630, CAM 5.2, RKSE 60, 1C 7, LL 002 and RCK 108) were used to stain the cervical tissues. CAM 5.2 reacted with the immature metaplasia strongly and the mature metaplasia weakly. 1C 7 and LL 002 reacted with the mature metaplasia strongly, compared to their reactivities in immature metaplasia. These results supported the idea that when reserve cells transform into squamous metaplasia, keratin 8 ceases to be expressed, and the synthesis of keratins 13 and 14 is initiated. The expression of keratin 8 was increased up to 77% of CIN III, compared to 15% of CIN I and II. About 90% of the squamous cell carcinoma contained keratin 8. Based on this observation, we hypothesized that the CIN lesions with expression of keratin 8 are progressive into cervical cancer. With respect to the keratin expression of endocervical adenocarcinoma, keratins 8 and 19 were found in all the cases, although keratins 5, 10, 13 and 14 were not found. This result indicated that the endocervical adenocarcinoma develops from the columnar cells.

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The aging change of glycoconjugate synthesis in the mouse kidney was studied by light microscopic radioautography with ³H-glucosamine. The density of the silver grains was quantitatively analyzed over the renal corpuscles and uriniferous tubules in eight age groups from fetal day 19 to postnatal 1 year.
As the results, all the cell types of the renal corpuscle, proximal and distal tubule of the nephron were labeled. The differences along with aging and among respective portions of the nephron were distinctive. The grain density of these three portions was shown to be generally in the following order in perinatal periods, from late fetal to early suckling: proximal tubule > distal tubule > renal corpuscle. The patterns of aging change of the grain density were similar among these three portions, i. e., the density was high in perinatal periods and then decreased with aging. The difference in the grain density was most remarkably observed between the perinatal periods and postnatal aged periods. The age-related change was remarkable in the proximal tubule as compared with the distal tubule and renal corpuscle.
Glycoconjugate synthesis in mouse kidneywas shown to be more active in perinatal periods than postnatal aged periods, especially in the proximal tubule.

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At various stages of the life cycle of cells, the occurrence of DNA single-strand breaks (SSB) is a common event. In order to understand better the relationship of SSB to physiological states of cells, one requires the analysis of SSB at the level of individual cells by in situ nick translation (ISNT). In the principle of ISNT, the DNA strand with breaks is elongated in the presence of biotin-11-dUTP by E. coil DNA polymerase I at the nicked sites. The biotin moieties incorporated into the newly synthesized strand are visualized enzyme-immunohistochemically with horseradish peroxidase-labeled antibiotin antibody. In this article, I will firstly describe the details of optimization of the ISNT reaction for its full implementation, using nicked λ phage DNA, which was fixed onto nitrocellulose filters, as well as using fresh frozen sections of rat testis and small intestine. Subsequently, I would like to demonstrate the presence of two types of SSB, which can be practically distinguished by protease-dependency of the staining; one is readily detected by ISNT without any deproteination steps (protease-independent type), and the other requires the protease treatment to be detected by ISNT (protease-dependent type). In our case, the single-strand breaks of DNA observed in terminally differentiated cells as well as the cells undergoing necrosis were protease-independent type. On the other hand, the DNA breaks in the cells undergoing replicative DNA synthesis and apoptosis were regarded as the latter type. Consequently, ISNT should be regarded as a useful molecular histochemical tool to categorize naturally occurring SSB.

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