Ultrastructural immunocytochemistry of a Leydig cell enzime and sperm tail protein using antigen retrieval by microwave irradiation Authors Mariana P. Musse Héctor E. Chemes Abstract We report here a simple and fast protocol for enhanced immunogold electronmicroscopic localization of various antigens in biological materials embedded in acrylic resins. Adult rat Leydig cells separated by density gradients and washed human spermatozoa were used to study the subcellular localization of specific antigens and to test the influence of exposure to microwave irradiation on the immunoreactivity assessed by immunogold labeling on formaldehyde fixed, LR White embedded material. Ultrathin sections mounted on nickel grids and immersed in citrate buffer were exposed for brief periods to microwave irradiation at full power (800 W). Immunoelectron microscopy was achieved using primary antibodies raised against a smooth endoplasmic reticulum steroidogenic enzyme (3β Hidroxysteroid Dehydrogenase, 3β- HSD) and a cytoskeletal protein of the sperm fibrous sheath (AKAP4). The secondary antibody was a gold conjugated (15nm particles) anti-rabbit immunoglobulin. Control sections were incubated in non immune serum. Sections were briefly exposed to osmium tetraoxide and uranyl acetate or were observed without any further staining. Labeling was expressed as gold particle density per µm2. Background labeling was negligible. 3β-HSD specifically localized to the smooth endoplasmic reticulum of Leydig cells, while mitochondria and lipid droplets were negative. AKAP4, the main protein component of the fibrous sheath of the sperm tail typically localized to the fibers of the fibrous sheath, while other components of the sperm tail were negative. After on section exposure to microwaves, gold density over the Smooth Endoplasmic Reticulum of Leydig cells and the fibrous sheath of spermatozoa increased by three fold over the same tissues not pretreated by microwaves without compromising the specific localization of the immunolabeling. This method allows for a significant increase of sensitivity and labeling density without modifying antibody concentrations, specificity or background staining. Downloads Download data is not yet available. Downloads Text complet (Català) Published 2007-12-28 Issue 56, 2005 : Endocrinologia molecular / Jaume Reventós editor Section Articles License The intellectual property of the articles belongs to the respective authors. At the time of submitting the articles to Treballs de la Societat Catalana de Biologia, authors accept the following terms: — Authors assign to the SCB (a subsidiary of the Institut d’Estudis Catalans) the rights of reproduction, public communication (including communication through social networks) and distribution of the articles submitted for publication to Treballs de la Societat Catalana de Biologia, in any form and medium, including digital platforms. The Publications Committee reserves the right to accept or refuse submitted articles and the right to make any editorial changes it deems appropriate. If the suggested changes are accepted by authors, they should re-submit the article with such changes. — Authors answer to the SCB for the authorship and originality of submitted articles. In other words, authors assure that submitted articles do not contain fragments of works by other authors or fragments of their own previously published works; that the content of articles is original, and that the copyright of third parties is not infringed upon. Authors accept this responsibility and undertake to hold harmless the SCB for any loss or damage resulting from non-compliance with this obligation. Furthermore, they should include a statement in articles submitted to the journal regarding their responsibility for the content of the articles. — Authors are responsible for obtaining permission for the reproduction of all graphic material included in articles, and they should moreover ensure that images, videos, etc., have been created with the consent of the individuals appearing in them, and that material belonging to third parties is clearly identified and acknowledged as such within the text. Likewise, authors should provide the respective consents and authorisations to the SCB when submitting articles. — The SCB is exempt from any liability arising from the possible infringement of intellectual property rights by authors. In all cases, it undertakes to publish corrections, clarifications, retractions and apologies, if necessary. — Unless otherwise stated in the text or in the graphic material, the contents published in the journal are subject to an Attribution - NonCommercial - NoDerivs 3.0 Spain (by-nc-nd) license from Creative Commons, the full text of which may be consulted at https://creativecommons.org/licenses/by-nc-nd/3.0/es/deed.en. Therefore, the general public is authorised to reproduce, distribute and communicate articles as long as their authorship and publishing entity are acknowledged, and no commercial use is made of them nor derivative work produced from them. — The journal is not responsible for the ideas and opinions expressed by the authors of published articles.