In-situ hybridization of nodulin mRNAs in root nodules using non-radioactive probes

Abstract

In-situ hybridization of nodulin mRNAs in root nodules using non-radioactive probes.

Pinus taeda phenylpropenal double-bond reductase: Purification, cDNA cloning, heterologous expression in Escherichia coli, and subcellular localization in P. taeda

“…After rinsing with TE buffer, the tissue sections were incubated in hybridization buffer (minus probes) containing: 0.3 M NaCl, 10 mM Tris-HCl (pH 7.4), 1 mM EDTA, 1 · Denhardt's, 50% formamide, 0.25 mg ml À1 yeast tRNA (Sigma) and 10% dextran sulfate at 25°C for 2 h. Next, tissue sections were hybridized to the appropriate probes at equal concentration (2 and 5 lg ml À1 hybridization buffer) and incubated at 37°C for 12 h. Post-hybridization washes were conducted with increasing stringency from 4 · SSC to 2 · SSC 1 · SSC and 0.1 · SSC at 25°C for 30 min each to remove non-specific binding. Hybridized riboprobes were detected using indirect immunolocalization as follows: first, sections were rinsed with TBST (10 mM Tris-HCl, pH 7.5, 10 mM NaCl, 0.3% Tween 20) then blocked with TBST containing 1% BSA, 0.1% PVP and 0.5% whole sheep serum (BBI) at 25°C for 1 h and finally incubated in sheep-anti-DIG antibody (Roche) at 1:100 in TBST containing 1% BSA at 25°C for 2 h. Sections, washed in TBST, were then treated with the chromogenic substrates NBT (nitroblue tetrazolium chloride, Roche) and BCIP (5-bromo-4-chloro-3-indolyl-phosphate, 4-toludine blue, Roche) in Tris (0.1 M, pH 9.5) containing 10 mM NaCl, 50 mM MgCl 2 , and 0.24 mg ml À1 levamisol (Sigma) (Bochenek and Hirsch, 1990). After 30 min at 25°C, the reaction was stopped by incubating the sections in TE buffer (pH 8.0) for 10 min.…”

Different temporal and spatial expression of two hydrophobin-encoding genes of the edible mushroom Agaricus bisporus

“…The protocol used for in situ hybridization was a modification of the method used by Bochenek & Hirsch (1990). Fruit body tissue was fixed in 4 '/o paraformaldehyde, 0.25 '/o glutaraldehyde in 0.1 M potassium phosphate buffer (pH 7.4) and for embedding we used paraffin.…”

Analysis ofrecQGene Transcript in Fruiting Bodies of Basidiomycetous MushroomLentinula edodes

“…Treatment of the thin sections before hybridization, hybridization with the RNA probes, and washing of the hybridized sections were done basically as reported by Bochenek and Hirsch1. 17) Immunological detection of the hybridized probes was done with a digoxigenin-nucleic acid detection kit (Roche Diagnostics GmbH, Penzberg Germany). Since the level of transcription of the Le.recQ gene is relatively low probably owing to its promoter property (the Le.recQ promoter possesses only the TATA box-like sequence as a consensus sequence), 13) the hybridization signals given by the The same amount (1 mg each) of total cellular RNAs was used for the experiments.…”