5 bromo 4 chloro 3 indolyl phosphate

In situ hybridization was performed according to our previous report with some modifications^{66 (link)}. Digoxigenin-labelled RNA probes were prepared with the MAXIscript Kit (Thermo Fisher, Waltham, MA, USA), in accordance with the manufacturer’s instructions. The DNA fragment of CTGF (NM_010217, located between 2252 and 2372) was subcloned into the pGEM-T Easy vector (Promega, Madison, WI, USA) and used to generate sense and antisense probes. Sections were deparaffinized and incubated with 3 μg/ml proteinase K (Roche, Mannheim, Germany) for 15 min at 37 °C. After fixation and prehybridization, sections were hybridized overnight at 42 °C with digoxigenin-labelled RNA probes. RNase A treatment (20 μg/ml; Roche) was carried out at 37 °C for 30 min. The sections were incubated with 1.5% blocking reagent (Roche) for 60 min at room temperature and then with anti-digoxigenin antibody conjugated with alkaline phosphatase (Roche) for 40 min at room temperature. Nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate (Roche) were used for signal detection.

Digoxigenin (Dig)–labeled probes against Sema3B, Sema3F, Nrp1, and Nrp2 were used as previously described (68). Briefly, sections were fixed for 10 min in 4% PFA [in 1× PBS (pH 7.4)], permeabilized for 10 min in 0.2 M HCl, and acetylated in 0.1 M triethanolamine with 5 mM acetic anhydrid for 15 min. Hybridization was performed overnight at 56°C with a probe concentration of 3 ng/μl. Slides were blocked for 2 to 3 hours, using 2% blocking reagent (Roche), followed by the detection of the Dig-labeled riboprobe with an anti-DIG Fab fragment conjugated with AlkP (1:750; Roche). The colometric reaction was performed using a mixture of 5-bromo-4-chloro-3-indolyl phosphate (Roche) and Nitro blue tetrazolium chloride (Roche).

Grains of cv. Morex sampled at various developmental stages were fixed in 50% (v/v) ethanol, 5% (v/v) acetic acid and 3.7% (w/v) formaldehyde overnight at 4°C, dehydrated and embedded in paraffin. Cross‐sections (12 μm) were mounted on a silane‐coated slide (Sigma‐Aldrich, Darmstadt, Germany), and the preparations were de‐waxed, rehydrated and exposed to 2 μg ml⁻¹ proteinase K for 30 min at 37°C. Finally, the tissue sections were dehydrated in preparation for the hybridization/immunological detection procedures performed as described (Radchuk et al., 2006). The hybridization probe was 1 ng μl⁻¹ digoxigenin‐labeled either sense or antisense Jek3 RNA synthesized from cDNA using either T3 or T7 RNA polymerase (Roche, Mannheim, Germany). The sections were challenged with alkaline phosphatase‐conjugated anti‐digoxigenin antibody and the signal generated was visualized by providing 4‐nitroblue tetrazolium chloride and 5‐bromo‐4‐chloro‐3‐indolyl phosphate (Roche).

To examine proteins, whole-cell lysates were prepared by suspending 1 A₆₀₀ unit-equivalent of cells in 100 μl SDS-PAGE buffer (57 (link)). SDS-PAGE samples were incubated at 100 °C for 10 min, prior to electrophoresis (tris-glycine, 10% (w/v) acrylamide). Proteins were stained with Coomassie Brilliant Blue R-250. For immunoblotting, protein samples were transferred to nitrocellulose membranes (Amersham Protran, 0.45 μm). Primary antibodies were murine monoclonal anti-His₅ (Qiagen; diluted 1:3000) and secondary antibodies were either horseradish peroxidase–conjugated goat anti-mouse IgG (Qiagen; diluted 1:3000). Detection employed horseradish peroxidase–substrate Luminata Classico (Millipore). To analyze polysaccharides in whole-cell lysates, samples were prepared as above and then incubated with 50 μg proteinase K for 1 h at 55 °C. The lysates were then separated by SDS-PAGE and transferred to PVDF (Amersham HyBond P 0.45 μm) or nylon membranes (BioDyne B; Pall). Membranes were probed with murine monoclonal antigen antibody P2B1G2/A9 ((58 (link)) diluted 1:350), followed by alkaline phosphatase–conjugated goat anti-mouse secondary antibody (Qiagen; diluted 1:3000). Colorimetric detection employed nitro-blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate (Roche).