Sodium borohydride

To determine the distribution of LepRb expression in the mouse brain, we performed single-label free-floating ISHH on wildtype mice brain tissue (n = 4). The mouse LepRb-specific riboprobe (a generous gift from Dr. Christian Bjørbæk) was generated using reverse-transcriptase polymerase chain reaction (RT-PCR) of mouse whole brain RNA (Ambion, Austin, TX) and the RT-PCR kit (Advantage; Stratagene, La Jolla, CA). The following primers were used to amplify a 400-base pair fragment corresponding to the intracellular domain of the long-form isoform of the leptin receptor: primer 1: 5′-AAAGAGCTCCTTCTCTGGGTCTCAGAGCAC-3′ and primer 2: 5′-AAAAAGCTTCTCACCAGTCAAAAGCACACCAC-3′ where the single-underlined sequence represent restriction enzyme recognition sites (primer 1: SacI site and primer 2: HinDIII site) and the double-underlined sequences are complementary to the mouse LepRb. The resulting PCR products were digested with SacI and HinDIII restriction enzymes and cloned into the pGEM-11Zf (+) plasmid (Promega, Madison, WI). Inserts were confirmed by DNA sequencing using T7 and Sp6 primers, Sequenase (Amersham Life Sciences, Arlington Heights, IL), and the ³⁵Sequetide system (New England Nuclear, Boston, MA). Plasmids were linearized with HinDIII or SacI for generation of sense and antisense probes, respectively. These linearized templates were transcribed in vitro into cRNA using either T7 or Sp6 polymerase (Ambion).
The in situ hybridization procedure was a modification of that previously reported by our laboratory (Priestley et al., 1993; Elias, 1998 (link); Yamamoto, 2003; Liu, 2003 (link)). Tissue was rinsed with DEPC-treated PBS, pH 7.0, for 1 hour before being pretreated with 1% sodium borohydride (Sigma) in DEPC-PBS for 15 minutes. After washing in DEPC-PBS, tissue was briefly rinsed in 0.1M TEA, pH 8.0, and incubated for 10 minutes in 0.25% acetic anhydride in 0.1M TEA. The tissue was then rinsed in DEPC-treated 2× SSC before hybridization. cRNA probes were diluted to 10⁶ cpm/mL in 50% formamide, 10 mM Tris-HCl, pH 8.0 (Gibco-BRL, Bethesda, MD), 5 mg tRNA (Invitrogen, Carlsbad, CA), 10 mM dithiothreitol, 10% dextran sulfate, 0.3M NaCl, 1 mM EDTA, pH 8.0, and 1 × Denhardt’s solution (Sigma) and applied to the tissue. Sections were incubated overnight at 57°C. Tissue was rinsed four times in 4× sodium chloride/sodium citrate (SSC) before being incubated in 0.002% RNase A (Roche Applied Bioscience, Indianapolis, IN) diluted in 0.5M NaCl, 10 mM Tris-HCl, pH 8.0, and 1 mM EDTA (RNase buffer) for 30 minutes at 37°C. After 30 minutes in RNase buffer, and two rinses at room temperature in 2× SSC, tissue was washed three times in 50% formamide in 0.2× SSC for 10 minutes at 50°C. Tissue was then washed 2× SSC at 50°C, 0.27× SSC at 55°C, and 0.2× SSC at 60°C for 1 hour at each wash. After rinsing twice in 2× SSC at room temperature, tissue was mounted on SuperFrost Plus slides (Fisher, Pittsburgh, PA), dehydrated in 3-minute incubations of increasing concentrations of ethanol (50%, 70%, 80%, 95%, and 100%), and delipidated for 5 minutes in chloroform. After washes in 100% and 95% ethanol, tissue was air-dried and slides were placed in X-ray film cassettes with BMR-2 film (Kodak, Rochester, NY) for 2 days. Slides were then dipped in NTB2 photographic emulsion (Kodak), dried, and stored in desiccant-containing, foil-wrapped slide boxes at 4°C for 4 weeks. Slides were developed with D-19 developer, counterstained with thionin, and dehydrated in an increasing concentration of ethanol, cleared in xylenes, and coverslipped with Permaslip. A control experiment where an antisense LepRb riboprobe was used on tissue pretreated with RNase A (200 µg/ml) demonstrated the specificity of the protocol.