Pronase

After linearisation of plasmids (pGEM-3Z, Promega, Madison, Wisconsin, USA) containing specific sequences of the genes for hIL-1beta (R&D Systems, Minneapolis, USA) and hIL-1R type 1 and type 2 (kindly provided by Immunex, Seattle, WA, USA), 35S-labeled run-off anti-sense and sense (control-) transcripts were generated using Sp6 and T7 RNA polymerases (Gibco BRL). ISH for the detection of RNA transcripts was performed as previously described [2 (link)]. In brief, dewaxed and rehydrated paraffin sections were exposed to 0.2 N HCL and 0.125 mg/ml pronase (Boehringer, Mannheim, Germany) followed by acetylation with 0.1 M triethanolamine pH 8.0/0.25% (v/v) acetic anhydride and dehydration through graded ethanols. Slides were hybridized to 2–4 x 10⁵ cpm of labeled probes overnight at 54°C. Washing and autoradiography was performed as described [2 (link)]. All sections were processed in parallel using the same batches of reagents and probes. The incubation of sections with Micrococcus nuclease (Boehringer Mannheim, Mannheim, Germany) prior to in situ hybridization resulted in the extinction of the specific autoradiographic signal, establishing that RNA sequences were the targets of the hybridization procedure. ISH signals were semiquantitated by counting the proportion of positive HRS cells and estimating the density of silver grains as the correlate for the transcript levels.

Spermidine-tetrahydrochloride (SPD), guinea pig liver TG2 (GPL-TG2), standard crystallins from bovine eye lens, trichloroacetic acid (TCA), dithiothreitol (DTT), ortho-phthalaldehyde (OPA), dansylchloride, trifluoroacetic acid (TFA), acetonitrile (C₂H₃CN), MDL 72527 (N,N′-Bis(2,3-butadienyl)-1,4-butanediamine dihydrochloride) and all reagents were from Sigma-Aldrich (St. Louis, MO, USA). [³H]-SPD (18 Ci/mmol) were obtained from NEN Radiochemicals (Italy). N¹-mono(γ-glutamyl)SPD, N⁸-mono(γ-glutamyl)SPD, N¹-N⁸bis-(γ-glutamyl)SPD were generously provided by J. E. Folk (National Institutes of Health, Bethesda, MD, USA). Pronase, collagenase, aminopeptidase M, carboxypeptidases A, B, and Y were obtained from Boehringer, (Mannheim, Germany). Usual laboratory chemicals were purchased from Merck (Darmstadt, Germany).