CEDIE   05498
CENTRO DE INVESTIGACIONES ENDOCRINOLOGICAS "DR. CESAR BERGADA"
Unidad Ejecutora - UE
capítulos de libros
Título:
Ultrastructural Immunocytochemistry of a Leydig Cell Enzime and a Sperm Tail Protein Using Antigen Retrieval by Microwave Irradiation
Autor/es:
MUSSE M.; CHEMES H.E.
Libro:
Proceedings of the CSB
Editorial:
Societat Catalana de Biologia
Referencias:
Lugar: Barcelona; Año: 2005; p. 101 - 106
Resumen:
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 (3b Hidroxysteroid Dehydrogenase, 3b-HSD) and a cytoskeletal protein of the sperm fibrous sheath (AKAP4). The secondary antibody was a gold conjugated (15 nm 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 mm2. Background labeling was negligible. 3b-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.