Postembedding immunogold staining was used to quantify low numbers of intracytoplasmic cytokine interferon molecules in cells embedded in Lowicryl K4M by the rapid technique of Altman et al. (1984). When labelling with colloidal gold-protein A (CGPA) was performed as suggested by the manufacturers, the relatively high background precluded the statistically significant distinction of small amounts of specific label. The standard procedure of postembedding staining was modified by introducing: (1) UV-irradiation of the sections before immuno-staining; (2) 10- to 50-fold greater dilution of CGPA than recommended by the manufacturers, and (3) washing the sections at 60 degrees C between staining steps. The resulting decrease in nonspecific binding of CGPA facilitated the quantification of as few as 6-10 CGPA particles per 1,000 micron2 of the surface of the Lowicryl K4M sections at greater than a 95% confidence level.
Download full-text PDF |
Source |
|---|
Publication Analysis
Top Keywords
Similar Publications
The precise subcellular localization of Dlg in the Drosophila larva body wall using improved pre-embedding immuno-EM.
J Neurosci Res
March 2018
Medical School Southeast University, Nanjing, China.
Discs-large (Dlg) plays important roles in nerve tissue and epithelial tissue in Drosophila. However, the precise positioning of Dlg in the neuromuscular junction remains to be confirmed using an optimized labeling method. In this study, we improved the method of pre-embedding immunogold electron microscopy without the osmic tetroxide procedure, and we found that Lowicryl K M resin and low temperature helped to preserve the authenticity of the labeling signal with relatively good contrast.
View Article and Find Full Text PDFThe sleeping beauty kissed awake: new methods in electron microscopy to study cellular membranes.
Biochem J
March 2017
Ultrapole, Ultra-Structural Bio-Imaging, Institut Pasteur, 28, rue du Dr. Roux, Paris 75015, France
Electron microscopy (EM) for biological samples, developed in the 1940-1950s, changed our conception about the architecture of eukaryotic cells. It was followed by a period where EM applied to cell biology had seemingly fallen asleep, even though new methods with important implications for modern EM were developed. Among these was the discovery that samples can be preserved by chemical fixation and most importantly by rapid freezing without the formation of crystalline ice, giving birth to the world of cryo-EM.
View Article and Find Full Text PDFPre- and Post-embedding Immunogold Labeling of Tissue Sections.
Methods Mol Biol
January 2018
School of Molecular Biosciences, BLS 202F, Washington State University, 1770 NE Stadium Way, Pullman, WA, 99164, USA.
Despite the improved resolution capacities of fluorescence microscopy over the last 20 years, localization of specific proteins at the ultrastructural level with gold-conjugated antibodies remains a valuable technique in the cell biological tool chest. Ultrastructural immunolocalization of specific proteins in tissues rather than in cultured cells is often advantageous because, in tissues, the interactions between different cell types and with the extracellular matrix are maintained. Here, we describe two immunogold labeling procedures to localize at the ultrastructural level one or more proteins.
View Article and Find Full Text PDFLabeling of ultrathin resin sections for correlative light and electron microscopy.
Methods Cell Biol
December 2012
Center for Regenerative Therapies, TU Dresden, Fetscherstraße 105, D-01307 Dresden, Saxony, Germany.
Correlative microscopy combines the versatility of the light microscope with the excellent spatial resolution of the electron microscope. Here, we describe fast and simple methods for correlative immunofluorescence and immunogold labeling on the very same ultrathin section. The protocols are demonstrated on sections of tissue samples embedded in the methacrylate Lowicryl K4M.
View Article and Find Full Text PDFPresence of F-actin in sperm head of Armadillidium peraccae (Isopoda, Oniscidea).
Tissue Cell
October 2011
Dipartimento di Biologia Marcello La Greca, Via Androne 81, Università di Catania, 95124 Catania, Italy.
Sperm of Armadillidium peraccae have been examined with cytochemical and immunocytochemical methods for fluorescence and electron microscopic visualization of cytoskeleton components. Sperm incubation in an antibody anti-β-tubulin shows only the presence of two centrioles located in the cytoplasmic region above the nucleus; no other microtubules are present in the sperm head. Instead, fluorescence microscopy of sperm incubated in FITC-phalloidin allowed to detect the presence of a large amount of F-actin in the apical region of the sperm head.
View Article and Find Full Text PDFWant AI Summaries of new PubMed Abstracts delivered to your In-box?
Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!