We report on the immunohistochemical demonstration of an enzyme at the electron microscopic level using specimens processed by rapid fteezing and the freeze substitution technique without the use of any chemical fixatives. Fresh rat liver tissue blocks were rapidly frozen by the metal contact method using liquid nitrogen, and were freeze-substituted with acetone without any chemical fixatives at-8O C. Some ofthe freeze-substituted tissues were embedded in Lowicryl K4M at-20'C; the others were returned to room temperature and embedded in Epok 812 at 60C. Ultra-thin sections were stained using anti-peroxisomal catalase antibody by the protein A-gold technique. The ultrastructure of the hepa

We examined the immunohistochemical distribution of the two mammalian isoforms of calcineurin catalyic subunits, A alpha and A beta, in central nervous system (CNS) tissues of cows, rats, and humans. Cryostat sections and paraffin sections of parformaldehyde-fixed tissues were stained with antipeptide antibodies for each isoform. The same localization pattern was observed in both cryostat and paraffin sections. In the latter, the intensity of the staining was dramatically enhanced by microwave irradiation. Calcineurin isoforms were localized in a variety of nerve cells but not in neuroglial cells. Their differential expression as the A alpha isoform in the nucleus and the A beta isoform in the cytoplasm was present in a variety of CNS nerve cells, most distinctively in Purkinje cells of the cerebellum and pyramidal cells of the cerebrum, irrespective of species. These results suggest that each isform has distinct intracellular sites of action in CNS neurons and that the phenomenon has been conserved during mammalian evolution.

Abstract. The kidney tissues of ddY mice from fetal to postnatal day 14 were labeled in vitro with either 8H-thymidine or 8H-uridine and analyzed by light and electron microscopic radioautography. The labeling index (L.I.) with 8H-thymidine or grain-number-per-cell with 3H-uridine was calculated by light microscopic radioautography. The L.I. of glomeruli and uriniferous tubules in the metanephric cortex were high throughout the embryonic stages, and decreased rapidly after birth. Many 3H-thymidine labeled immature cells were observed in the embryonic stages by electron microscopic radioautography. The grain-number-per-cell of glomeruli and uriniferous tubules labeled with 3H-uridine decreased gradually from fetal to postnatal stages.

(3A3157).We report compositional changes in glpconjugates in mouse kidney cortex due to aging, as analyzed by lectin histoche-mistry and Western blot analysis. Mouse kidaey tissues ofprenatal and postnatal ages (prenatal, 19 days of gestation; postnatal 2 and 8 days, 4 and 13 weeks, and 10 months) were fued in 4% paraformaldehyde and cryosections were made.Tbey were stained with 16 kinds of biotinylated lectin, fol-

S U M M A R YWe studied the localization of calcineurin by immunoblotting analysis and immunohistochemistry as a first step in clarifying the role of calcineurin in the retina. Rat, bovine, and human retinal tissues were examined with subtype-nonspecific and subtypespecific antibodies for the A ␣ and A ␤ isoforms of its catalytic subunit. In mature retinas of the three species, calcineurin was localized mainly in the cell bodies of ganglion cells and the cells in the inner nuclear layer, in which amacrine cells were distinctively positive. The calcineurin A ␣ and A ␤ isoforms were differentially localized in the nucleus and the cytoplasm of the ganglion cell, respectively. Calcineurin was also present in developing rat retinas, in which the ganglion cells were consistently positive for it. The presence of calcineurin across mammalian species and regardless of age shown in the present study may reflect its importance in visual function and retinal development, although its function in the retina has not yet been clarified.

Peroxisomes, since their discovery as microbodies, have been studied mostly independently by electron microscopists and biochemists. The fine structure has been studied by electron microscopy, and the compositional enzymes and proteins by protein biochemistry. Electron microscopic histochemistry has been used to try to clarify the relationship between the fine structure and its constituents. The immunogold technique, a combination of electron microscopy and protein biochemistry, for the first time resolved this problem due to the high sensitivity and resolution power of the staining and the high reliability of the technique. The present paper reviews the way in which the immunogold techniques, especially the protein A-gold technique, revealed the localization of various enzymes or proteins in peroxisomes or peroxisomal subcompartments, and discusses why this technique should be employed in peroxisome research.

No abstract

We report a change in the proliferative activity of mouse colonic epithelium due to development and aging. In order to measure the proliferative activity, colonic epithelium was immunostained for cyclin proliferating cell nuclear antigen (PCNA/cyclin), which appears from the Gl to the S phase of the cell cycle, and compared with labeling obtained by [3H]-thymidine radioautography. Litter mice of six age groups from the fetal period (embryonic day 19), newborn period (postnatal day 1), suckling period (postnatal day 5), weaning period (postnatal dy 21), adult period (2 month old) to the senescent period (11 month old) were examined by immunohistochemistry. The descending colons were fixed in methacarn (method-Carnoy) and embedded in paraffin. Sections were stained for PCNA/cyclin activity using 19A2 monoclonal antibody and the avidin-biotin peroxidase complex (ABC) technique. For radioautography, litter mice of nine age groups using in vivo intraperitoneal administration of [3H]-thymidine. The labeling indices of colonic epithelial cells in the proliferative zone were then analyzed and compared between the two investigative methods. Our results show that the proliferative activity of mice colon was high in the fetal and newborn periods and almost constant from the suckling period to senescence, as demonstrated by both PCNA/cyclin immunohistochemistry and [3H]-thymidine radioautography. The labeling index seen by PCNA/cyclin immunohistochemistry was, however, higher than that seen by [3H]-thymidine radioautography.