In situ hybridization at the electron microscope level: localization of transcripts on ultrathin sections of Lowicryl K4M-embedded tissue using biotinylated probes and protein A-gold complexes.

Abstract

Abstract.A technique has been developed for localizing hybrids formed in situ on semi-thin and ultrathin sections of Lowicryl K4M-embedded tissue. Biotinylated dUTP (Bio-11-dUTP and/or Bio-16-dUTP) was incorporated into mitochondrial rDNA and small nuclear U1 probes by nick-translation. The probes were hybridized to sections of Drosophila ovaries and subsequently detected with an anti-biotin antibody and protein A-gold complex. On semi-thin sections, probe detection was achieved by amplification steps with anti-protein A antibody and protein A-gold with subsequent silver enhancement. At the electron microscope level, specific labeling was obtained over structures known to be the site of expression of the appropriate genes (i.e., either over mitochondria or over nuclei). The labeling pattern at the light microscope level (semi-thin sections) was consistent with that obtained at the electron microscope level. The described nonradioactive procedures for hybrid detection on Lowicryl K4M-embedded tissue sections offer several advantages: (a) rapid signal detection; (b) superior morphological preservation and spatial resolution; and (c) signal-to-noise ratios equivalent to radiolabeling.

Post-embedding in situ hybridization for localization of viral nucleic acid in ultra-thin sections.

“…The temperature and duration of hybridization used were much higher and longer than those reported by other authors (Troxler et al, 1990;Binder et al, 1986;Lawrence and Singer, 1985). A temperature of 65°C is close to that used for heat denaturation; therefore, improved signals could be obtained.…”

“…In addition to ease of sample preparation, the post-embedding methods can prevent the loss of structural morphology or selective extraction of cell components that may occur during the pre-embedding process when detergents or proteases are applied. Satisfactory results have been obtained with plastic sections of Lowicryl-embedded samples directly for in situ hybridization (Guellec et al, 1991;Troxler et al, 1990;Thiry and Thiry-Blaise, 1989;Binder et al, 1986). The main disadvantage of Lowicryl as an embedding material is that only nonosmicated tissues can be embedded.…”

The Intracellular Distribution of Influenza Virus Matrix Protein and Nucleoprotein in Infected Cells and Their Relationship to Haemagglutinin in the Plasma Membrane

“…It is not known whether viral RNA is present within the dense nuclear structures. However, the technology to answer this question is now available since in situ hybridization can be performed on Lowicryl sections (Binder et al, 1986).…”

Double labelling in situ hybridization using non-isotopic and isotopic detection.

“…A detailed description of the protocols, will be included in Singer et al (submitted for publication) . A different approach to ultrastructural detection has been published by Binder et al (1).…”