The procedure for non-radioactive in situ hybridization was described previously 19 (link), 21 (link). Briefly, total RNA was extracted from DM3 tooth germs at E40-E60. The degenerate primers for Wnt3a, Wnt5a, Dkk1, β-Catenin, Axin2, Lef1 and Tcf4 are listed in Table S1 . After reverse transcriptase-polymerase chain reaction (RT-PCR), the correct-sized bands were extracted from agarose gels and their DNA sequences were determined. The RNA probe was synthesized using digoxigenin-UTP with T7 RNA polymerase according to the manufacturer's protocol (DIG RNA labeling Mix, Roche). For the staining procedure, slides were first rehydrated and then treated with proteinase K (1 μg/ml in PBS) for 30 min at 37 °C, and re-fixed with 4% paraformaldehyde. The specimens were then dehydrated in series of ethanol (25, 50, 75 and 100%). After drying in air for 1 h, the specimens were hybridized at 70 °C overnight. After 3-4 hours of rinsing with SSC solution, specimens were incubated with alkaline phosphatase conjugated anti-digoxigenin Fab (Roche) overnight. Signals were detected with NBT/BCIP substrates (Promega).
Alkaline phosphatase conjugated anti digoxigenin fab
Manufactured by Roche
Alkaline phosphatase-conjugated anti-digoxigenin Fab is a lab equipment product that serves as a detection agent. It is used to identify and quantify the presence of digoxigenin, a chemical compound, in various samples.
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7 protocols using alkaline phosphatase conjugated anti digoxigenin fab
1
Non-radioactive In Situ Hybridization Protocol
2
In Situ Hybridization Analysis of Tooth Germ Development
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The procedure for in situ hybridization was described previously 20 (link). Briefly, RT-PCR was performed using mRNA from tooth germ of miniature pigs. The correct size bands were extracted from agarose gels and DNA sequencing was performed. The RNA probe was synthesized by in vitro transcription according to the protocol of DIG RNA labeling Mix (Roche). For the staining procedure, after serial rehydration, the slides were treated with proteinase K (1 μg/ml in PBS) for 30 min at 37°C. After being re-fixed with 4% paraformaldehyde, the sections were dehydrated in series of ethanol (25, 50, 75 and 100%). After being dried for 1 h, the specimens were hybridized with probe at 70°C overnight. After being washed for hours, the sections were incubated with alkaline phosphatase conjugated anti-digoxigenin Fab (Roche) overnight. Signals were detected with NBT/BCIP substrates (Promega). Primers used for RT-PCR were list as follows:
WNT5a (forward: 5'-ctggcaggactttctcaagg-3';
reverse: 5'-cgcgctgtcatacttctcct-3');
β-Catenin (forward: 5'-ggtccatcagctttccaaaa-3';
reverse: 5'-ctgaacaagggtcccaagaa-3');
Axin2 (forward: 5'-gagggagaaatgcgtggata-3';
reverse: 5'-tgggtgagagtttgcacttg-3').
3
Immunohistochemistry and In Situ Hybridization for Skin Tissue
4
Immunohistochemistry and In Situ Hybridization for Skin Tissue
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For section immunostaining and in situ hybridization, fixed skin tissue was embedded in paraffin and sectioned at 6–7 μm. After de-paraffination, sections were processed for immunohistochemistry or in situ hybridization. The MITF antibody was from Abcam (ab12039, 1:200 dilution). The peroxidase staining was used after primary antibody treatment as described (Jiang & Chuong, 1992 (link)). Non-radioactive in situ hybridization was performed as described (Chuong et al., 1996 (link)). Briefly, the sections were treated with proteinase K (10 μg/ml in PBS) for 20 min, re-fixed with 0.2% glutaraldehyde/4% paraformaldehyde, and rinsed with PBT. The sections were then prehybridized in hybridization buffer (containing 50% formamide, 5× sodium citrate/sodium chloride buffer, 1% sodium dodecyl sulfate, 50 μg/ml heparin, 50 μg/ml tRNA) at 65°C for 1 hr. After prehybridization, sections were placed in new prehybridization buffer containing 1–3 μg/ml digoxigenin-labeled riboprobes and hybridized overnight at 65°C. Finally, sections were incubated with alkaline phosphatase-conjugated anti-digoxigenin Fab (Roche, Indianapolis, IN) overnight. Positive signals were detected by incubating the specimens with NBT (nitro-blue tetrazolium)/BCIP (5-b romo-4-chloro-3′-indolyphosphate) substrates (Promega, Madison).
5
Non-Radioactive In Situ Hybridization Protocol
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The procedure for non-radioactive in situ hybridization has been described previously (Wang et al., 2017 (link)). Briefly, RT-PCR was performed using mRNA from tooth germ of WZSPs. The correct size bands were extracted from agarose gels and DNA sequencing was performed. The primers used for pig Pitx2, Msx2, Left, Dlx2 were listed in Table S1 . The RNA probe was made by labeling with digoxigenin-UTP by in vitro transcription with T7 RNA polymerase according to the protocol of DIG RNA labeling Mix (Roche). For the staining procedure, mandible samples were rinsed in RNAse-free PBS and fixed in 4% paraformaldehyde in PBS (pH 7.5). The fixed tissues were decalcified, embedded in paraffin and cut into slices (6 μm). After the rehydration, the slides were treated with proteinase K (1 μg ml−1 in PBS) for 30 min at 37°C, and then re-fixed with 4% paraformaldehyde in PBS then rinsed with PBS. The specimens were then dehydrated with series of ethanol, before leaving the slides to air dry for 1 h. The specimens were hybridized in hybridization buffer at 70°C overnight. After washing for 3–4 h, specimens were incubated with alkaline phosphatase conjugated anti-digoxigenin Fab (Roche) overnight. Positive signals were detected by incubating the specimens with NBT/BCIP substrates (Promega).
6
In Situ Hybridization of Slc17a5 and Virma
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The total RNA was extracted from submandibular glands or spleens of C57BL/6 mice. The degenerate primers used for Slc17a5 and Virma are listed in Supplementary Table 6 . After RT-PCR, the correct-sized bands were extracted from agarose gels, and their DNA sequences were determined. The RNA probe was synthesized using digoxigenin-UTP with T7 RNA polymerase (Roche) according to the manufacturer’s protocol (DIG RNA labeling Mix, Roche). For the staining procedure, slides were first rehydrated, then treated with proteinase K (1 μg/ml in PBS) for 30 min at 37 °C, and finally refixed with 4% paraformaldehyde. The fixed specimens were dehydrated in a series of increasing ethanol concentrations (30, 50, 75, 90, and 100%). After drying in air for 1 h, the specimens were hybridized at 70 °C overnight. After 3−4 h of rinsing with SSC solution, specimens were incubated with alkaline phosphatase-conjugated anti-digoxigenin Fab (Roche) overnight. Signals were detected with NBT/BCIP substrates (Promega).
7
In situ PCR for Gene Expression
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In situ PCR was conducted as described below. The roots were sliced into 50 μm-thick sections using Microtome (Leica, Germany). Then the samples were transferred into 100 μl sterile water with RNase inhibitor (1 U per μl), added 8 U DNase and incubated at 25 °C for 20 min to eliminate the genomic DNA, and then stop the reaction by adding 15 mM EDTA and heating to 75 °C for 10 min. The cDNA were synthesis with gene-specific primers (ZmNSA1-cDNA for ZmNSA1 and Zm18S-cDNA for 18S ribosomal RNA; Supplementary Data 2 ), then PCR amplifications were conducted in a reaction system containing 1 × PCR buffer, 1.5 mM MgCl2, 200 μM dNTPs, 0.4 nM digoxigenin-11-dUTP (Roche), 0.5 μM primers and 2 U Taq DNA polymerase (Thermo Fisher, USA). Following the PCR amplification, the samples were washed twice for 5 min with PBS buffer, blocked for 30 min in 0.1% BSA, incubated for 1 h with 1.5 U alkaline phosphatase-conjugated anti-digoxigenin Fab (Roche), washed twice for 15 min with washing buffer (0.1 M Tris-HCl, 0.15 M NaCl, pH9.5), stained with BM Purple AP Substrate precipitating (Roche) for 40 min, then washed twice with water and photographed using a Olympus microscope (BX53). The primers and sequences were listed in Supplementary Data 2 .
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