Monitoring morphology and signal during non-radioactive in situ hybridization procedures by reflection-contrast microscopy and transmission electron microscopy.

Abstract

We analyzed the effects of steps in RNA in situ hybridization (ISH) procedures on morphology and hybridization signal with reflection-contrast microscopy (RCM) and transmission electron microscopy (TEM). In chessboard experiments, a range of fixatives containing formaldehyde, glutaraldehyde, or both, and various permeabilization protocols, including ethanol and pepsin treatment, were investigated. A transfected rat fibroblast cell line that harbors an inducible human cytomegalovirus immediate early (IE) transcription unit, and specific probes for 28S ribosomal RNA and IE messenger RNA were used for this purpose. Probes were labeled with digoxigenin and hybrids were detected with anti-digoxigenin F(ab)2 fragments conjugated to horseradish peroxidase, followed by diaminobenzidine/H2O2 reaction. Effects of fixation and pre-treatments on RNA detection efficiency and morphology were monitored by RCM on whole cells. After Epon embedding and ultra-thin cross-sectioning, the corresponding TEM images were obtained. With the pre-treatments analyzed, it appeared impossible to find an acceptable balance between ISH signals and preservation of ultrastructural morphology: when good signal-to-noise ratios are obtained, the ultrastructural morphology is already deteriorated. We discuss the parameters that influence the fragile balance between high RNA detection efficiency and good preservation of ultrastructure and the benefit of RCM monitoring in the development and procedures for pre-embedding electron microscopic ISH.

Simultaneous restitution of matrix and cells in gastric ulcer: use of a combined in-situ hybridization and immunohistochemistry technique applicable to paraffin-embedded tissue

“…These groups have used animal sera to block nonspecific binding of antibodies to tissue specimens; in contrast, by using bovine serum albumin instead of sera, Hermanson et al (1994) have been able to perform immunohistochemistry prior to ISH with no appreciable loss of signal or unacceptable background staining. The need to permeabilize sections with proteolytic pretreatment for ISH may result in a loss of the epitope of interest for immunohistochemistry (Macville et al 1995). Consequently, execution of immunohistochemistry prior to ISH seems attractive to us.…”

Applications of reflection‐contrast microscopy, including the sensitive detection of the results ofin situhybridisation a review

“…0nly slightly adjusted (EM) methods are needed for producing RCM-(EM) specimens. Macville et al (1995) also studied RCM in combination with (EM). They reported that (EM) morphology could be predicted and extrapolated to a large extent from the RCM images.…”

Detection and amplification systems for sensitive, multiple-target DNA and RNA in situ hybridization: looking inside cells with a spectrum of colors

“…Especially in cases where difficulties arise with recording of faint staining of, for example, small nucleic acid target sequences, RCM detection is preferable over brightfield detection. Initial ISH studies used the reflection properties of the HRP-DAB reaction product and resulted in the efficient visualization of DNA targets, ranging from repetitive to single-copy sequences (10-40 kb), and mRNA sequences (Landegent et al 1985;Ambros et al 1986;Cornelese-Ten Velde et al 1988;Multhaupt et al 1989;MacVille et al 1995). The application of other enzyme precipitation reactions for RCM detection, however, was hampered by the fact that most reaction products are only stable in aqueous-based mounting media (see Table 5) and, thus, are unstable in immersion oil, which is required in this procedure.…”