隆肛蛙卵巢类固醇激素受体的免疫细胞化学研究

用免疫细胞化学方法,对隆肛蛙不同发育时期卵泡雌激素受体、孕激素受体和雄激素受体进行了定位检测。结果表明,雌激素受体和孕激素受体主要在卵黄合成和卵黄合成后期的滤泡细胞中表达;3种激素受体在各期卵母细胞胞质中均有表达,雌激素受体和孕激素受体在卵黄合成和卵黄合成后期表达强烈;3种类固醇激素受体在卵母细胞核膜中都有表达。这些结果说明,在隆肛蛙卵母细胞生长发育和成熟中,雌激素受体和孕激素受体主要调控卵黄合成及合成后期雌二醇和孕酮的合成与分泌,其受体可能通过基因组和非基因组两种机制发挥作用。     

Localization of calcium regulatory hormones in fish

Immunocytochemical localization of hypocalcin, a hypocalcemic factor in the corpuscles of Stannius (CS), in American eels was examined at the light (ABC method) and electron microscopic (protein A-gold technique) levels with the specific antiserum raised against purified rainbow trout hypocalcin. Only type 1 cells in the CS were immunoreactive in the light microscopic immunocytochemistry. At the electron microscopic level, however, hypocalcin immunoreactivity was observed in secretory granules of both type 1 and type 2 cells. Our findings may indicate that type 1 cells are the main source of hypocalcin, but that type 2 cells also produce it, suggesting that the presence of two cell types reflects different physiological conditions of a single cell type, rather than functionally different cell types. In addition, we summarize our recent data on the localization of other calcium regulatory, or putative calcium regulatory, hormones in fish: parathyroid hormone, calcitonin and calcitonin gene-related peptide.     

Universal antisera for immunocytochemical identification of two different gonadotrophs in acanthopterygian fishes

Pituitaries of various teleosts belonging to 25 orders were immunostained with antisera raised against synthetic fragment peptides corresponding to conservative regions of gonadotropin subunits (mummichog FSH 50-60 and mummichog LH 91-106). Both immunoreactive FSH cells and immunoreactive LH cells were successfully identified in the fishes of almost every order of the superorder Acanthopterygii and several species of the superorders Paracanthopterygii and Polymixiomorpha, such as mullet, alfonsino, flyingfish, mackerel, flounder, cod, beardfish, etc. These antisera are therefore considered as universal antisera for immunocytochemical application to acanthopterygian fishes. Extensive diversity in the abundance of the FSH cells and the LH cells among species was noted even in fishes with similar gonadal stages, indicating the possibility that the respective roles of FSH and LH may vary considerably among species in advanced teleosts.Evident but generally weak immunoreactivities to anti-mummichog LH 91-106 were observed in the fishes of the superorder Cyclosquamata; and slight or weak immunoreactivities to the antiserum were observed in the fishes of several more primitive taxa (superorder Stenopterygii, Protacanthopterygii, Ostariophysi, subdivision Clupeomorpha, and subdivision Elopomorpha). No immunoreactivity to anti-mummichog FSH 50-60 was observed in these fishes. These results are consistent with the phylogenetic status of the fishes and the degree of conservativeness in the amino acid sequences of the antigen regions.     

Biotransformation enzyme activities in the olfactory organ of rainbow trout (Oncorhynchus mykiss). Immunocytochemical localization of cytochrome P4501A1 and its induction by β-naphthoflavone

Olfaction is a crucial function in most fish species, but little is known about biotransformation enzymes in the olfactory organ. This study demonstrates that biotransformation enzymes usually found in the rainbow trout liver, are present in the olfactory organ as well. While microsomal cytochrome P450 reductase, p-nitrophenol hydroxylase and cytosolic glutathioneS-transferase presented similar levels in both the olfactory organ and the liver, microsomal 7-ethoxyresorufinO-deethylase (EROD), 7-ethoxycoumarinO-deethylase, and 7-pentoxyresorufinO-deethylase were much lower in the olfactory organ (77-, 35-, 200-times respectively). Furthermore, microsomes from the olfactory organ were able to perform testosterone hydroxylation only in the 16α-position while testosterone was hydroxylated in the 16β-position by liver microsomes. Using polyclonal antibodies raised against perch cytochrome P4501A1, the immunoreactive protein was shown to be strongly expressed in various cellular types forming the nonsensory epithelium. Some immunostaining was also reported in the nonsensory cellular elements constituting the sensory epithelium, while olfactory receptor cells failed to show cytochrome P4501A1-immunoreactivity. Finally, the exposure of rainbow trout to waterborne β-naphthoflavone (0.1 μg ml⁻¹) for 2 or 4 days resulted in a higher induction of EROD activity in the olfactory organ compared to the liver. The presence of biotransformation enzymes in the olfactory organ of rainbow trout addresses the question of their involvement in the detoxication/toxication of pollutants as well as in the olfactory function.     

Monoclonal Antibody Production and Immunolocalization of a Salinity Stress-Related Protein in Rice (Oryza sativa)

Among various physiological responses to salt stress, the synthesis of a lectin-related protein of 14.5 kDa was observed in rice plants (Oryza sativa L.) under the treatment of 170 mmol/L NaCl. In order to better understand the role of the SALT protein in the physiological processes involving salinity, it was immunolocalized in mesophilic cells of leaf sheath and blade of a rice variety IAC-4440 following monoclonal antibodies produced by hybridome culture technique. This variety turned out to be an excellent model for that purpose, since it accumulates SALT protein even in absence of salt treatment and it has been classified as moderately sensitive to salinity and a superior grain producer. This feature was relevant for this work since it allowed the use of plants without the deleterious effects caused by salinity. Immunocytochemistry assays revealed that the SALT protein is located in the stroma of chloroplasts under non-stressing condition. Since the chloroplast is the main target affected by salinity and considering that the SALT protein does not present any apparent signal peptide for organelle localization, its lectin-like activity seems to play an important role in the establishment of stable complexes, either to other proteins or to oligosaccharides that are translocated to the chloroplast.     

SD大鼠肾小管上皮细胞两种原代培养及传代方法的比较

目的：建立较理想的大鼠肾小管上皮细胞原代培养、传代及鉴定方法。方法：采用肾小管节段贴块法及0．2％胰蛋白酶消化20min两种方法进行原代培养,以0．25％胰蛋白酶(A组)、0．125％胰蛋白酶-0．02％EDTA(B组)消化传代,利用免疫细胞化学方法鉴定细胞种类。结果：两种方法均能成功培养肾小管上皮细胞,但前者较好,小管节段贴壁早。B组成功传代(4代)并鉴定为肾小管上皮细胞,A组传代失败。结论：肾小管节段贴块、0．125％胰蛋白酶-0．02％EDTA消化是大鼠肾小管上皮细胞原代培养及传代的有效方法。     

鱼类消化道内分泌细胞概述及研究方法

消化道内分泌细胞能产生多种具有调节胃肠功能的胃肠激素。该类激素除了能促进胃肠对营养物质的消化与吸收外,还能控制摄食行为、调控消化道运动以及细胞营养作用,甚至能够影响其他一些内分泌腺的活动。了解肠道内分泌细胞的基本概况有助于深入地研究鱼类的消化生理,组织化学、免疫细胞化学和电子显微镜等技术是研究消化道内分泌细胞鉴别、定位以及形态学的重要方法,利用这些技术能够揭示鱼类消化道内分泌细胞的生物功能及作用机理,对研究鱼类摄食、消化和吸收等生理机制有重要意义。因此,本文综述了近年来国内外鱼类消化道内分泌细胞的研究进展及主要研究手段,旨在为鱼类消化生理学、内分泌学和营养学提供参考资料。     

鱼类消化道内分泌细胞概述及研究方法

消化道内分泌细胞能产生多种具有调节胃肠功能的胃肠激素。该类激素除了能促进胃肠对营养物质的消化与吸收外,还能控制摄食行为、调控消化道运动以及细胞营养作用,甚至能够影响其他一些内分泌腺的活动。了解肠道内分泌细胞的基本概况有助于深入地研究鱼类的消化生理,组织化学、免疫细胞化学和电子显微镜等技术是研究消化道内分泌细胞鉴别、定位...     

Optimizing methods in immunocytochemistry: one laboratory's experience

Abstract: The addition of immunocytochemical staining procedures to a diagnostic cytology service enables greater specificity of interpretation for many common disease conditions, especially neoplastic diseases. However, well‐tested immunohistochemical techniques may require modification for cytologic specimens, and other considerations are necessary when working with air‐dried cells. In this article, we describe our experience in evaluating options for sample transport and handling, and discuss methods for obtaining control cells from a variety of tissues for use in immunocytochemical staining. Important immunocytochemical principles and techniques, including fixation, antigen retrieval, and use of primary and secondary antibodies in manual and automated staining systems are described as used in our laboratory for cytologic specimens. Although we emphasize methods relevant to diagnostic laboratories receiving samples from external clients, the information is also applicable to any laboratory interested in adding or enhancing immunocytochemical services.