Histological changes of the mammary tissues during the growth of pregnancy-dependent mammary tumors (PDMT) and the immunohistochemical staining of epidermal growth factor (EGF) in these mammary epithelial cells were studied in the inbred strain of GR/A mice. Mice were mated at 60-70 days of age and subjected to concurrent pregnancy. Mice developing PDMT at the first pregnancy always retained the microscopically detectable tumors during the subsequent inter- or early pregnancy when no gorss PDMT existed. EGF staining was observed in the epithelial cells of the lactating mammary glands showing marked proliferation, but not in PDMT. These findings demonstrate that in GR/A mice PDMT repeat growth and regression, but not the complete extinction, during the reproduction and suggest that the contribution of EGF to the growth of PDMT is rather minor.

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The localization of metallothionein (MT), a heavy metal binding protein of low molecular weight, in the brain of young and old macaca fascicularis was investigated by immunohistochemical technique, and the amounts of MT and heavy metals (Zn, Cu, Cd) were measured by radioimmunoassay and atomic absorption spectrophotometer, respectively. Immunohistochemically, MT was found in pia mater, ependymal cells, protoplasmic astrocytes of the gray matter and fibrous astrocytes of the white matter of cerebrum. Cytoplasm and processes of protoplasmic and fibrous astrocytes showed strong MT immunostaining. Also, vascular feet and adventitia gave positive MT immunostaining. Moreover, both protoplasmic and fibrous astrocytes in pons and spinal cord showed the same positive MT immunostaining as those in cerebrum. In the cerebellum, Bergmann's glias, protoplasmic astrocytes of granular layer and nucleus dentatus, and fibrous astrocytes of white matter showed a strongly positive immunostain for MT.
According to the radioimmunoassay, the amount of MT in the brain of the old one was relatively high-about 19.3 μg/g wet weight. For the heavy metals in the brain, zinc and copper were detected, excepting cadmium.

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The localization of metallothionein (MT), a heavy metal binding protein of low molecular weight, in the human brain was investigated by the immunohistochemical technique, and also the amounts of MT were assayed by radioimmunoassay. Immunohistochemically, MT was found in pia mater, ependymal cells, protoplasmic astrocytes and neuropil of the gray matter, and fibrous astrocytes of the white matter of the brain. Cytoplasm, processes and some nuclei of protoplasmic and fibrous astrocytes showed strong MT immunostaining. Vascular feet and adventitia of the blood vessels were also positive for MT immunostaining. With aging, MT deposited thickly in the pia mater and in the dilated perivascular spaces showing reticular appearance, especially in those, so-called etat crible of basal ganglia and centrum semiovale of hypertensive and aged cases. According to the radioimmunoassay, the amount of MT in the brain increased with age.

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The three-dimensional computer images of the alignment of collagen III fibrils at epithelial-mesen-chymal interfaces during critical stages of submandibular gland morphogenesis in the 12- to 13-day-old mouse embryos were reconstructed from serial paraffin sections stained with an anti-collagen III antibody. The computer program used for processing section data was Cosmozone 2S, which required only a personal computer and a coordinate digitizer interfaced to the computer graphics system. The mid 12-day gland with a round lobule showed a rather randomly scattered distribution of the collagen on the surfaces of the lobule and stalk. The interface at the joining portion of the lobule and stalk showed a significant accumula-tion. In the late 12-day gland with signs of cleft initiation, a specific accumulation of the collagen was clearly seen on basal epithelial surface in shallow clefts. This tendency was much more evident as cleft formation proceeded. The images reconstructed from both frontal and transverse section data of the 13-day gland indicated that epithelial surface of the cleft was surrounded by continuous collagen bundles, suggesting the involvement of the collagen during the epithelial branching.

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The immunoreactivity of chromogranin was studied in postnatal salivary glands of the mice and rats. Chromogranin was observed in proacinar cells in both sexes during the postnatal developmental stage of the submandibular glands; the strongest reactivity was seen in 4th to 7th day specimens of mouse, and in 7th to 21st day specimens of rat. Acinar cells of mouse parotid glands showed positive staining in 1 to 42 days. No chromogranin staining was seen in granular convoluted tubule (GCT) cells of the submandibular glands in mice and rats. In sublingual glands of rat, reactivity of chromogranin was found in undifferentiated serous acinar cells, whereas in mice, it was absent in these cells. No sex difference of chromogranin staining was observed in submandibular glands in mice and rats. Immunoblotting of chromogranin using polyclonal anti-bovine SP-1/chromogranin antiserum showed in submandibular glands and adrenal glands of the rat consisted of chromogranin A, B and C. The results of this study suggest that the existence of chromogranin in proacinar cells of salivary glands is probably associated with the developmental phenomenon during the postnatal development.

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We examined the suitability of the freeze-substitution and paraffin embedding technique for the im-munolocalization of membrane-bound and soluble antigens in the rat at the light microscopic level. Frozen tissues were substituted at -80°C in methanol or acetone containg chemical fixatives, i. e., formaldehyde, glutaraldehyde, acrolein, hexamethylene diisocyanate (HMD), diethyl malonimide (DEM) and 1-ethyl-3- (3-dimethyl-aminopropyl) carbodiimide (water soluble carbodiimide, WSC). The following antigens were localized using monoclonal antibodies and antisera: A) membrane-bound antigens; γ-glutamyl transpeptidase (γ-GTP), common antigen of secretory granule membrane (SG 170 antigen) and Golgi associated antigen (GAA 108); and B) soluble antigens; branched-chain amino acid transferase type I isozyme (BAT), glutamate dehydrogenase (GDH), pancreatic amylase, proliferating cell nuclear antigen (PCNA), rat serum albumin and IgG.
1) The freeze-substitution technique maintained an excellent tissue structure and conservation of antigenicity. By using this method, BAT was localized in mitochondria in iver cells, and γ-GTP was demonstrated in the secretory granule membrane of pancreatic acinar cells, although conventional fixation methods provided negative reaction. 2) In general, the membrane-bound antigens were localized in detail with a strong immunoreaction in the tissues substituted in solvent alone; however, for the localization of soluble antigens, tissues substituted in solvents containing chemical fixatives revealed a strong and precise antigen localization. 3) Formaldehyde and glutaraldehyde proved to be better fixatives concerning the conservation of structure and antigenicity than the other chemical reagents. 4) The choice of substitution solvent was important for the immunohistochemistry of some antigens. For example, methanol was suitable for PCNA, and substitution in acetone was essential for γ-GTP using one of the monoclonal antibodies to γ-GTP.

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Vascular endothelial cells (ECs) have been demonstrated to play crucial roles in inflammation and immune responses by expressing various adhesion molecules, such as MHC class II antigens, ICAM-1 and ELAM-1 (E-selectin), and by synthesizing various cystokines including IL-1, IFN-γ and TNF. They act as antigen presenting cells in leading to a cascade of immunologic and inflammatory events. The expression of cell adhesion molecules by ECs, particularly high endothelial venules, mediates permeation of lymphocytes and leucocytes in inflammatory lesions, and subsequently leads to continuous inflammation. Both morphological and functional alterations of ECs named “activation” are induced by cytokines in vitro and inflammatory or immune responses, and the concept of endothelial activation is useful in understanding endothelial cell functions. In addition, the immunoperoxidase technique is useful for studying morphological and functional changes of ECs in inflammatory and immune diseases.

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In order to examine sex steroid hormone receptor expression in the central and peripheral tissues relative to reproduction, we have analysed estrogen (ER) and progestin receptors (PR) and their mRNAs in rat brain, and human uterine and other tissues, using immunocytochemical assay (ICA), reverse transcrip-tion-polymerase chain reaction (RT-PCR) and in situ hybridization technique (ISH) Summary and conclusion are as follows;
1) ICA studies confirmed nuclear localization of ER or PR in human uterus and ovarian endometriosis. Good correlation exists between the ER-ICA and ligand binding assay (LH20 assay) or ER-enzyme immunoassay (EIA), indicating the usefulness of the ER-ICA for the semiquantitative measurement in humanuterine tissues. The ER score of ovarian endometriotic tissue was much less than that of the normal endometrium, suggesting much lower hormonal responsiveness in the pathologic tissue.
2) ERmRNA analysis:
(1) Northern blot analysis using rat ERcRNA probe demonstrated 6.6 kb ERmRNA in rat brain (the hypothalamus-preoptic area, HPOA), anterior hypophysis (AP), uterus, tube, ovary, and testis. Additional subspecies of 4.2, 2.6 and 2.0 kb were identified in the testis.
(2) A very sensitive and specific RT-PCR assay for ERmRNA was developed to analyse the receptor ex-pression in more detail. The RT-PCR product 287 bp was generated from tissue RNA using the primer set derived from the rat ERcDNA sequence and its authenticity was confirmed by direct sequencing. Quan-tification of the RT-PCR assay was made possible by measurement of the hybridization signals in a Bio-Im-age Analysing System, BAS-2000. A standard curve was obtained from graded dilutions of the uterine RT-PCR products.
The levels of ERmRNA were as follows; AP>HPOA, amygdala (AMY) >cerebral cortex (CC), cerebellum (Ce). The existence of a small amount of ERmRNA in the “non-target” CC and Ce implied adirect action of estrogen on these brain regions.
3) PRmRNA analysis:
In analogous experiments as ERmRNA, a very sensitive and specific RT-PCR assay for PRmRNA was also developed to detect and quantify PRcDNA in the tissues. Since rat PRcDNA has been not cloned and sequenced, rat PRmRNA was synthesized with the primer set derived from the human PRcDNA sequence. The RT-PCR product of 320 bp was generated from tissue RNA, and the authenticity of the rat uterine product was confirmed by direct sequencing.
The levels of PRmRNA in adult rat brain were as follows; AP, HPOA, AMY, CC>Ce, with smaller differential distribution than that of ERmRNA, and roughly paralled the levels of PR with exception of the CC where the PR level is low. The expression mechanism of PR in the CC may be different from that in the other brain regions.
The early postnatal development of PRmRNA was found. The increment of the cerebral PRmRNA might be associated with the early drastic increase of PR in the neonatal rat brain cortex.
4) ER-and PRmRNAs analysis in human uterus:
(1) Primary structure of the uterine ERmRNA, amplified by the RT-PCR and determined by direct sequencing of the product, showed that ERmRNA expressed in the normal human endometrium has Gly-400 (GGG) and Thr-594 (ACA).
(2) Northern blot analysis of ER-and PRmRNAs using riboprobes demonstrated greater message levels in the endometrium than the myometrium, respectively. The results were in agreement with estrogendependent induction of PR.
5) In situ hybridization studies.
We have detected ER- and PRmRNAs and mapped out the distribution of the meassages in the intact female rat brain using rat ER- and PRcRNA synthetized. The distribution of both mRNAs was similar, with higher density in the arcuate nucleus and the ventrolateral part of the ventromedial nucleus of hypothalamus, indicating a possible coexpression in the same neurons.

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A method to localize glucocorticoid receptor (GR) by utilizing the specific interaction between GR and glucocorticoid responsive element (GRE) is described. In this study, pBR 322 DNA was thymine-thymine (T-T) dimerized by UV-irradiation and used as a probe, since pBR 322 DNA harbors GRE DNA consensus sequences and it is known that GR binds to pBR 322 DNA specifically. T-T dimerized pBR 322 DNA was incubated with fresh frozen sections of adrenalectomized rat liver, which were fixed in 4% paraformaldehyde in phosphate buffered saline, and the sites of reaction were visualized by a successive use of rabbit anti-T-T antibody and horseradish-peroxidase labeled goat anti-rabbit IgG antibody. As a result, GR was localized to the nuclei and nuclear membranes as well as cytoplasm of hepatocytes, and most of the staining was lost in the liver sections from rats, which were intraperitoneally injected with hydrocortisone. On the other hand, the staining for T-T dimerized DNA probe with cyclic AMP responsive element consensus sequence was localized solely to the nuclei and the staining pattern was not altered by an injection of the steroid, indicating that the staining for pBR 322 DNA is sequence-specific. Finally, this method should provide useful information on the localization of steroid hormone receptors with DNA-binding activity.

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Localization of fat/muscle-type glucose transporter (GLUT4) was examined in rat soleus muscle by immunofluorescence and immunogold labeling of frozen sections. Semithin and ultrathin frozen sections were stained with anti-peptide antibodies raised against the C-terminus of GLUT4. In the muscle sections from untreated and fasted animals, GLUT4 was concentrated intracellularly in the trans-Golgi and the trans-Golgi reticulum regions. GLUT4 was also found in vesicles and tubules near the plasma membrane. Insulin administration resulted in the appearance of the GLUT4 reactivity at the plasma membrane, while a significant amount of GLUT4 remained intracellularly in vesicles and tubules and also in the Golgi. These observations suggest that vesicles and tubules near the plasma membrane and in the trans-Golgi regions are reservoirs of intracellular GLUT4 and that fusion of these vesicles and tubules with the plasma membrane upon insulin stimulation is the mechanism to supply the plasma membrane with GLUT4.

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It is of increasing interest to observe the precise localization of cyclic 3′, 5′-nucleotide phosphodiesterase (PDEase) activity in rod outer segments at the ultrastructural level. The cytochemical method of PDEase localization at the ultrastructural level was developed by Florendo et al. However, there were two major difficulties in the use of snake venom as an exogenous 5′-Nucleotidase (5′-Nase): 1) the marked ultrastructural damage due to snake venom, and 2) the inadequate penetration of exogenous 5′-Nase into tissues. Therefore, two improvements for the demonstration of PDEase activity were carried out by the case of: 1) a purified 5′-Nase as exogenous 5′-Nase for a better preservation of cell morphology, and 2) 40μm sections made with the use of a freezing microtome for a better penetration of 5′-Nase into the tissues.
PDEase activity using purified 5′-Nase was observed along the disc in the rod outer segment, and no retinal detachment was observed between the outer segments and pigment epithelium, the ultrastructure being excellently preserved, as compared with the use of snake venom. By applying this improved method to freeze-substitution technique, the catalytic site of PDEase was localized on the cytoplasmic surface of the disc membranes. This new technique would be useful in detecting the precise localization of PDEase activity.

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We report the immunohistochemical localization of protein kinase C isozymes (types I, II, III and ζ) in various organs using the monospecific monoclonal antibodies: MC-la, MC-2a and MC-3a, and a polyclonal antibody for a synthetic polypeptide of ζ, reviewing results reported previously (2-4, 20, 21, 23), and discussing the technical aspect of immunohistochemistry on protein kinase C. For light microscopy, tissues were fixed and cryo-sections or paraffin sections were made. They were stained by PAP, ABC and immunofluorescent technique. For immuno-electrom microscopy, pre-embedding immunoperoxidase and post-embedding immuno-gold technique were done. By light microscopic analysis, tissue specific differential localization of isozymes was observed. Three isozymes (types I, II and III) were consistently localized in the cytoplasm of various kinds of cell. On the contrary, ζ-related protein was located in the nucleus of Purkinje cells. The increased staining for protein kinase C isozymes was observed in human thyroid cancer tissues and cultured glioma cells. By immuno-electron microscopy, diffuse localization of protein kinase C (type III) in the cytoplasm of rod bipolar cells of the retina was observed. Type II isozyme was densely localized in the hyaluromere of blood platelets, and translocated to the cell membrane by TPA administration. The immunohistochemical techniques were very effective in elucidating the tissue specific localization of protein kinase C isozymes and their pathological changes.

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