Anti digoxigenin antibody conjugated with alkaline phosphatase

Prickle1^+/+ or C57BL/6 mice were used to analyze gene expression. The Prickle1 probe [39 (link)] for in situ hybridization was generated by in vitro transcription from the plasmid and then labeled with digoxigenin. Dissected cochleae were digested with 20 mg/ml of Proteinase K (Ambion, Austin, TX, USA) for 20 min, and then hybridized overnight at 60°C to the riboprobe in hybridization solution containing 50% (v/v) formamide, 50% (v/v) saline sodium citrate and 6% (w/v) dextran sulfate. After washing off the unbound probe, the samples were incubated overnight with an anti-digoxigenin antibody conjugated with alkaline phosphatase (Roche Diagnostics GmbH, Mannheim, Germany). The samples were reacted with nitroblue phosphate/5-bromo, 4-chloro, 3-indolil phosphate (BM purple substrate, Roche Diagnostics GmbH, Mannheim, Germany), which changed the color to purple by alkaline phosphatase. The reaction was stopped by 4% PFA. Samples were then mounted in glycerol and viewed in a Leica M205 FA microscope. Images were captured with Nikon E800 compound microscope using Metamorph software. At least three mice were used for either Prickle1 or Vangl2 at any of the stages.

All tree shrews were anesthetized and perfused with 4% PFA. Their lungs were removed, dissected, and sectioned into 30-μm cross-sections. To label both sense and antisense rRNA probes for ISH, tree shrew CXCL10 cDNA was subcloned into the pGEM T-vector (Promega, USA). rRNA probes for hybridization were prepared by DIG RNA Labeling Mix (Roche, USA). The ISH was performed per standard methods: Briefly, tissue sections were hybridized with rRNA probes at 60 °C overnight. Sections were incubated with anti-digoxigenin antibody conjugated with alkaline phosphatase (Roche, USA). The ISH signals were further developed with 75 μg/ml of nitroblue tetrazolium and 175 μg/ml of 5-bromo-4-chloro-3-indolyl phosphate substrate (Solarbio, China).

Each probe’s cDNA template was amplified with the specific primers (listed in Table 2) and cloned into a pGEM-T plasmid (Promega, United Sates). Digoxigenin-labeled anti-sense or sense cRNA probes were transcribed in vitro using a digoxigenin RNA labeling kit (Roche Applied Science, Switzerland). In situ hybridization was performed as previously described (Hu et al., 2008 (link)). In brief, uteri were frozen sectioned into 10 mm, and then fixed in 4% (wt/vol) paraformaldehyde, permeabilized, and hybridized with each anti-sense cRNA probe (1:100) at 55°C overnight. A digoxigenin-labeled sense probe was used as the negative control. Following hybridization and post-hybridization washes, sections were incubated in an anti-digoxigenin antibody conjugated with alkaline phosphatase at 4°C overnight (Roche Applied Science, Switzerland). The signal was visualized as dark brown by incubating within a substrate solution containing 5-bromo-4-chloro-3-indolyl phosphate (Amresco, United Sates) and nitro blue tetrazolium (Amresco, United Sates). The activity of endogenous alkaline phosphatase was blocked by levamisole (2 mM, Sigma, United Sates). The sections were counterstained with 1% methyl green.

Digoxigenin-labelled RNA probes were prepared with the MAXIscript Kit (Thermo Fisher Scientific, Waltham, MA, USA), in accordance with the manufacturer’s instructions. The DNA fragment of Fgf4 (NM_010202, located between 117 and 731) was amplified by polymerase chain reaction (PCR), using primers shown in Supplementary Table S1. The fragment was subcloned into the pGEM-T vector (Promega, Madison, WI, USA) and used to generate sense and antisense probes. Bioengineered tooth germs after 5-day organ culture were fixed in 4% PFA overnight at 4 °C, treated with 6% H₂O₂ and 0.1% Tween 20 in PBS at room temperature, and incubated with 10 µg/mL proteinase K (Roche, Mannheim, Germany) for 3 min at 30 °C. After post-fixation and prehybridization, samples were hybridized overnight at 70 °C with digoxigenin-labelled RNA probes. Samples were blocked with 10% normal sheep serum (Sigma) and then incubated with anti-digoxigenin antibody conjugated with alkaline phosphatase (Roche), overnight at 4 °C. Nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate (Roche) were used for signal detection.

In situ hybridization was performed as described previously [75] (link), with the following modifications. Briefly, antisense RNA probes were generated from cDNA-containing plasmids and labeled with digoxigenin by in vitro transcription, using the DIG RNA labeling kit (Roche Applied Science, Cat# 11175025910). Whole-mount in situ hybridization was performed on mutant and corresponding control cochleae at various ages (P0, P3, P8, and P10). The 2% paraformaldehyde–fixed cochleae were digested briefly with 10 mg/mL of proteinase K for 5–10 min. Cochleae were hybridized with the Fgf8 probe overnight at 60°C in the hybridization solution. After washing, cochleae were incubated overnight at 4°C with an anti-digoxigenin antibody conjugated with alkaline phosphatase (Roche Applied Science, Cat# 11093274910). After several washes, cochleae were incubated in the dark with NBT/BCIP (BM purple substrate, Roche Applied Science, Cat# 11442074001). After periodic monitoring, the reaction was stopped by washing several times with KTBT. The organs of Corti were flat mounted and imaged.