In situ detection of specific gene expression during and immediately after transcription at electron microscopic level

Abstract

In situ hybridization (ISH) is a widely applied technique used for visualizing specific nucleic acid sequences at chromosomal, cytologic, and histologic levels. It sometimes fails, however, to demonstrate precise cell identity, early stages of gene expression and variants of alternative splicing because of its limited resolution. To overcome this shortcoming, we have developed an improved ISH technique at the electron microscopic (EM) level by conducting en bloc hybridization before embedding (pre-embedding) and immuno-EM detection after ultra-thin sectioning (post-embedding). We applied this technique to demonstrate both the dynamic expression of interleukin (IL)-6 mRNA immediately after lipopolysaccharide (LPS) treatment, and the static expression of osteonectin mRNA in a differentiating osteoblastic cell linage. Tissue samples were diced into 1 mm cubes, fixed with 4% paraformaldehyde, and then successively hybridized en bloc with the digoxigenin (DIG)-labeled single-stranded probe measuring 200–300 bp with the aid of microwave treatment. After washing, for EM observation, the cubes were embedded in epon for ultra-thin sectioning, and a gold-colloid-labeled anti-DIG antibody was used for post-embedding immuno-EM; some of the cubes was directly incubated with anti-DIG antibody and developed en bloc for stereoscopic and light microscopic observation. IL-6 mRNA during and immediately after transcription was demonstrated in the nuclei of the alveolar macrophages and in neutrophils of mouse lung tissue as early as 15 min after LPS treatment, which was of better sensitivity than that by Northern blot or nuclear run-on techniques. Moreover, in mouse calvaria tissue, osteonectin mRNA both in the nucleus and the cytoplasm was observed in a differentiating osteoblastic cell linage in a differentiation-specific manner. This technique is useful in identifying specific cell types during and immediately after transcribing specific mRNA based on ultrastructural morphology.