A single protocol to detect transcripts of various types and expression levels in neural tissue and cultured cells: in situ hybridization using digoxigenin-labelled cRNA probes

Abstract

We have developed a simple non-radioactive in situ hybridization procedure for tissue sections and cultured cells using digoxigenin-labelled cRNA probes. This protocol can be applied for the detection of various transcripts present at a wide range of expression levels in the central nervous system. Cerebellar hybridization signals for transcripts estimated to be expressed at high (MBP, myelin basic protein), moderate (GluR1, subunit of AMPA/kainate sensitive glutamate receptors) and low (inositol polyphosphate-5-phosphatase) levels of abundance are demonstrated as examples. The sensitivity and cellular resolution were significantly improved by avoiding any ethanol treatment commonly used in other procedures. The localization of a labelled cell with respect to its environment is shown to be more easily assessed by counterstaining of the tissue with the nuclear dye Hoechst 33258. The present protocol can be combined with immunocytochemistry as demonstrated for glial fibrillary acidic protein (GFAP). All steps of the procedure, including preparation and labelling of the cRNA probes, pretreatment of tissue, hybridization and visualization of the labelled transcripts, are described in detail.

Dynamin 2 homozygous mutation in humans with a lethal congenital syndrome

“…16 Dnm2 was expressed in most tissues, including the peripheral nervous system (Figure 3a, right panels), although no expression was evident in skeletal muscle or heart (for tissue profiles see Supplementary Table 3). Injection of one cell stage zebrafish embryos with a translational blocking morpholino (MO) targeting dnm2 transcripts resulted in pleiotropic phenotypes in dnm2 morphants: lethality was observed in 10% and bent-tail in 20% after 48 h post-fertilization (hpf; Supplementary Figure 5).…”

MDGA1, an IgSF molecule containing a MAM domain, heterophilically associates with axon- and muscle-associated binding partners through distinct structural domains

“…The detailed protocol is available upon request. The cDNAs of the subtracted library were isolated, cloned, and analyzed by in situ hybridization performed as described (Schaeren-Wiemers and Gerfin-Moser, 1993;Tsuchida et al, 1994). The cDNA clones that were expressed selectively by LMCm motor neurons were used to isolate longer cDNAs to identify their coding sequences.…”

Amelioration of chronic neuropathic pain after partial nerve injury by adeno-associated viral (AAV) vector-mediated over-expression of BDNF in the rat spinal cord

“…To enhance tissue penetration and avoid nonspecific background, the cRNA probes were adjusted to an average length of 100-200 bases by alkaline hydrolysis for 20 min at 60°C before use. 71 Riboprobes were quantified with a known amount of control DIG-labeled RNA and utilizing Roche's DIG Quantification Teststrips and DIG Wash & Block Buffer Set, following the manufacturer's recommendations.…”