Biomax mr film

In situ hybridization was performed according to general methodology, as described previously (Julien et al., 2008 (link), 2009 (link); Delay et al., 2014 (link)). DNA oligonucleotides were labeled with ³³P-dATP (PerkinElmer) using a three-terminal deoxynucleotidyl transferase enzyme kit (New England Biolabs). The reaction was conducted at 37°C for 2 h, and labeled oligonucleotides were purified with the QIAquick Nucleotide Removal Kit (QIAGEN). We used three different oligonucleotide mixes to hybridize to specific regions in the murine Shank3 mRNA (NM_021423.4). The first mix corresponds to sequences located before the ankyrin domain (nucleotides 337-296, 413-372, and 483-440); the second mix corresponds to sequences within the ankyrin domain (nucleotides 653-609, 1106-1061, and 1254-1209, which are deleted in the Shank3^Δex4-9 allele); the third mix corresponds to sequences after the ankyrin domain, within the proline-rich domain (nucleotides 3484-3440, 3693-3649, and 4127-4081). Prehybridation and hybridization conditions were performed as described by Julien et al. (2009) (link). Hybridized, 12 µm brain sections were exposed to Kodak Biomax MR films for 20 d. Nonspecific hybridization was considered negligible, as determined by adding a 100-fold excess of unlabeled probes.

Brains were rapidly removed and frozen at −40°C, then stored at −80°C. Frozen brains were cut into 20-µm coronal sections to harvest the ventral and dorsal striatum areas at +1.7mm to −0.3mm against bregma (Paxinos and Watson, 2007 ). Serial sections were collected onto polysine-coated slides and stored at −80°C prior to autoradiography. The autoradiography procedure was based on Strazielle et al. (1998) (link) and conducted according to Dawson et al. (2014) (link). After preincubation in 0.05 M sodium phosphate buffer (NaPB) pH 7.4, sections were treated with 20 pM [¹²⁵I]RTI-121 in the NaPB with increasing concentrations of 2-DPMP (0–30 µM) for 60 min at room temperature. Nonspecific binding was assessed in the presence of 200 µM nomifensine (control – “block”). Kodak BioMax MR films were applied over the rinsed and air-dried slides for three days. Autoradiograms were analyzed using MCID™, Version 7.0, Imaging Research Inc. (Interfocus Ltd, U.K.). Flat-field correction was applied. Each drug concentration was tested in six brains. The dorsal striatal (caudate-putamen, CPu) and accumbens (NAc) regions of interest (ROIs) were sampled in duplicates for relative optical density, left and right ROI values were averaged and their means were calculated to assess the specific binding.

GLP-1 receptor autoradiography was used to quantify the GLP-1 receptors in the mouse tumors in the presence or absence of vatalanib treatment. The Rip1Tag2 mice were treated with vatalanib as described before for 4 days and sacrificed on day 5. During necropsy, the tumors were snap frozen on dry ice. Tumor sections (20-μm thick) were incubated for 2 h at ambient temperature in the presence of 32 pM [¹²⁵I]-GLP-1 (2,000 Ci/mmol). The incubation solution was 170 mM Tris-HCI buffer (pH 8.2) containing 1% bovine serum albumin, bacitracin (40 μg/ml) and MgCl₂ (10 mM) to inhibit endogenous proteases. Non-specific binding was determined by adding 100-nM solution of unlabeled GLP-1. The incubated sections were washed twice for 5 min in cold incubation buffer containing 0.25% bovine serum albumin, then in buffer alone, and dried quickly. Finally, the sections were apposed to Biomax MR films (Kodak, Rochester, NY, USA) and exposed for 1 week in X-ray cassettes.
In all the experiments, the autoradiograms were quantified using a computer-assisted image processing system [26 (link),27 (link)]. Tissue standards for iodinated compounds were used for this purpose.

Total protein extraction was realized using the “Qproteome mammalian protein prep kit” (Qiagen^®) according to the manufacturer’s protocol. Protein concentrations were measured and 40µg of proteins were loaded into an 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred into nitrocellulose membranes (Biorad^®). Membranes were blocked with 5% nonfat dry milk in PBS-0.5% Tween 20 (PBS-T) at room temperature for 1h, followed by overnight incubation at 4°C with primary antibodies against alpha-1 type I collagen (ColIA1), fibronectin, LRP5, GAPDH or β-actin (R&D systems^®). The membranes were washed 3 times with PBS-T and incubated 1h at room temperature with horseradish peroxidase-conjugated secondary antibodies (RnDsystems^®). After PBS-T washing, membranes were incubated for 1 min with the enhanced chemiluminescence detection solution (ECL) (Perkin Elmer^®) and signals were detected using Biomax MR^® Films (Kodak^®).

Briefly, total protein was separated using 7.5–12.5% SDS-PAGE and transferred to PVDF membrane (Millipore, USA). Membranes were blocked in 5% fat-free milk and incubated with primary antibodies (Supplementary Table S4) for overnight at 4 °C. After washing, membranes were incubated with secondary antibody (Supplementary Table S5) for 2 h at room temperature (RT). Bands were detected using an enhanced chemiluminescence kit (Bio-Rad, USA) and blots were exposured to Kodak Biomax MR Films.