Sheep anti dig antibody

A 516 bp Arhgef2 PCR product was generated with primers listed in S5 Table and subsequently cloned into a p-AL2-T vector. The RNA probe was generated by in vitro transcription. Chromogenic/fluorescence in situ hybridization was performed on 16 μm thick brain sections from E11/P0 mice post-fixed in 4% PFA for 15 min and permeabilized with Proteinase K for 2.5 min at RT. The reaction was stopped by applying 0.2% glycine in PBS 1x, followed by 20 min post fixation with 20% glutaraldehyde in 4% PFA. The sections were treated with the pre-hybridization buffer (50% formamide, 5x SSC pH 7.0, 2.5 M EDTA, 0.1% Tween-20, 0.15% CHAPS, 0.1 mg/ml Heparin, 100 μg/ml yeast tRNA, 50 μg/ml salmon sperm DNA, 1x Denhardt’s solution) at 65°C for 2 hours followed by overnight incubation with Arhgef2 RNA probes at 65°C. Sections were then treated with RNase A for 30 min at 37°C, subsequently washed with SSC pH 4.5 in 50% formamide at 65°C and then with KTBT (100 mM NaCl, 50 mM Tris–HCl pH 7.5, 10 mM KCl, 1% Triton X-100) at RT. Following blocking in 20% sheep serum and incubation with sheep anti-DIG antibody (1:1,000, Roche, Mannheim, Germany) at 4°C overnight, the sections were washed in KTBT and NTMT (50 mM NaCl, 100 mMTris–HCl pH 9.5, 50 mM MgCl_2, 0.5% Tween-20) for 1 hour at RT. Development was achieved through addition of the chromogenic NBT/BCIP substrate (1:50, Roche).

TUNEL assays were done as described (Arama and Steller, 2006 (link)) with some deviations. DIG-labelled nucleotides were detected with a sheep anti-DIG antibody (Roche) and a donkey biotinylated anti-sheep secondary antibody (Jackson ImmunoResearch). For signal amplification, embryos were incubated for 45′ with ABC reagents (Vector Laboratories), followed by a 5′ incubation with TSA Flourescein reagents (Perkin Elmer) diluted 1:50. Embryos were mounted in ProlongGold (Life Technologies).

Gluten‐derived product‐specific IgE was analysed by IgE‐capture ELISA as previously described,⁹, ¹⁵ with the exception, that serum samples were diluted in 3% (v/v) rabbit serum (Almeco, Esbjerg, Denmark; Un Glu) or 3% (w/v) skimmed milk powder (Sigma‐Aldrich; E Glu, and Ac Glu 1–3). Briefly, ELISA plates were coated with mouse anti‐rat IgE antibody (HDMAB‐123 HydriDomus, Nottingham, United Kingdom). Two‐fold serial dilutions of serum samples were incubated on plates to obtain antibody Log₂ titre values. Specific IgE antibodies were detected using digoxigenin (DIG)‐coupled gluten‐derived product and HRP‐labelled sheep anti‐DIG antibody (11 633 716 001, Roche Diagnostics GmbH, Mannheim, Germany) with TMB‐one as substrate.