Dig labeling kit

BAC filters were hybridized with probes labeled and detected with CDP-Star (GE Healthcare) or DIG-labeling kit (Roche). Positive signals were confirmed by direct PCR with probe-specific primers on the individual clones. BACs of true positives were column-purified from 5 ml cultures, and end-sequenced with M13 Forward and Reverse primers. Each end sequence is designed for a pair of specific primers, which was used to determine the relative locations of overlapping BACs. Primer pairs from both distal ends were selected to label probes for the next round of chromosomal walking. Inverse PCR (Sambrook and Russell 2001 ) and TAIL-PCR (Liu and Whittier 1995 (link)) were performed to identify flanking sequences when no additional BAC clones could be identified. GpMTD1 probes were the same as those used for the GpMTD1 DNA gel-blot analysis (Hamaji et al. 2009 (link)). The PCR product of GPLEUFATG and GPLEURTAA was labeled to screen for LEU1S alleles (Supplemental Material, Table S1). Two gametologs (WDR57 and DRG1) and their flanking regions, which were not obtained in the MT+ BAC assembly, were obtained by genomic PCR using specific primers based on the MT– alleles. The linkage of MT+ DRG1 was determined by recombination scores as described below. Abundant repetitive regions flanking the MT assembly prevented further chromosomal walking to connect directly to autosomal sequences.

In situ hybridization was performed for perichondrium cambium layer markers Lgals1 and cartilage markers Col2a1 and collagen type X alpha 1 Col10a1 in 3‐day‐old rat proximal tibias and in vitro chondrocyte pellets. DNA templates for riboprobe transcription were amplified by PCR from epiphyseal cDNA using custom‐designed primers (Table 1) containing a T7 (for sense probes) or Sp6 (for antisense probes) promoter.
PCR products were purified by agarose gel electrophoresis and QIAquick Gel Extraction Kit (QIAGEN, Hilden, Germany). Single‐stranded riboprobes for in situ hybridization were transcribed using DIG Labeling Kit (Roche Diagnostics, Mannheim, Germany) that incorporates a digoxigenin‐ (DIG‐) conjugated uracil every 20 to 25 nucleotides. Labeled RNA probes were purified using Micro Bio‐Spin 30 Columns (Bio‐Rad, Hercules, CA, USA) and quantified using a NanoDrop Spectrophotometer (Thermo Fisher Scientific).

TRAP gene expression was studied by in situ hybridization. A gene sequence for rat TRAP [28 (link)] was amplified by conventional PCR using cDNA from rat bone and oligonucleotide forward and reverse primers: rnTRAP.for 5′-ACGCCAATGACAAGAGGTTC-3′, rnTRAP.rev 5′-ACATAGCCCACACCGTTCTC-3′ (Life Technologies, Carlsbad, CA, USA) and cloned in a Dual Promoter TA Cloning Kit (Life Technologies). The cloned insert was sequenced to establish the orientation (Seqlab, Göttingen, Germany). A digoxigenin (DIG)–conjugated complementary RNA probe was synthesized using T7 or Sp6 polymerase to yield the probe in the sense or antisense direction (DIG-labeling kit; Roche Diagnostics, Oslo, Norway). Longitudinal sections from the tibial diaphysis (Ovx-D/sham) and femoral diaphysis (experimental rickets) were subjected to hybridization following our established protocol [29 (link)]. TRAP mRNA⁺ osteocytes were quantified in cortical bone within 4–10 mm from the proximal EMB by point counting in a squared grid. Three sections were examined from each animal and their means compared between the groups. Tibial diaphyses were examined twice with an interclass correlation of p < 0.001 and Cronbach’s alfa of 0.94. Staining of osteoclasts from the femoral metaphysis in healing for 72 h was used as a positive control. The sense probe did not show any staining.

Whole mount ISH was performed, essentially, as previously described [69 ] except BM purple (Roche) was used as the chromogenic substrate. Briefly, plasmids encoding cDNA for mouse Stat3, Scx, MyoG, or Sox9 were linearized and DIG-labeled complimentary probes were generated using a DIG-labeling kit (Roche). As indicated, probes were hybridized overnight and detected using alkaline phosphatase-labeled anti-DIG antibody with subsequent chromogenic substrate development. For sectioning, embryos stained as above were embedded into paraffin wax after clearing in CitriSolv (Fisher Scientific) and sectioned serially at 16μM by microtome.

According to the manufacturer’s instructions for DIG labeling KIT (Roche), we generated digoxigenin (dig)-labeled RNA sense and antisense probes by in vitro transcription of the cht5 cDNA clone obtained from Drosophila Genomics Resource Center (#1135722; FBcl0236609). Further genes/clones: cht2, LD28264; cht3, LP01426; cht6, IP07037; cht 7, LD 45559; cht12, AT18578; idgf1, RE68533; idgf3, RE62596; idgf6, LD34164. The fixed embryos were washed with PBT and then fixed again for 20 min with PBT: 4% formaldehyde (1:1). We washed embryos with PBT/hybridization buffer (1:1 ratio) three times and finally for 60 min at 60 °C. Hybridization occurred at 60 °C ON. We washed embryos intensively with PBT for standard antibody staining (procedure see above). The dig-labeled RNA was detected with an anti-Dig alkaline phosphatase-conjugated antibody (1:500; Fab fragment; sheep), which we visualized accordingly to the manufacturer’s instructions (Roche).

Riboprobes were generated using a DIG-labeling kit (Roche). Two templates were amplified from cDNA and cloned into pBS (KS). The 5′ template including the signal peptide sequence of nbr was amplified with primers (5′-ATGGTACCTCGCAATGAGTGATTTAC-3′) and (5′-TATGGATCCTGCAGTTGGTTCTCTAGT-3′) generating a 467 base pair probe. The 3′ template from the cytoplasmic part of the gene was amplified with the primers, (5′-ACAAGTCGTCGTACAAGGA-3′) and (5′-GACCACCATTCTTGTTTGTAGGCA-3′) generating a 343 base pair probe. The procedure for in situ hybridization was carried out according to [46 (link)]. A sense probe was used as a negative control.

Acr-Hox gene-specific primer design was performed with the sequence assembling software Geneious 6.1.6 (Biomatters Limited) and primers were purchased from Life Technologies Company (Thermo Fischer Scientific). First strand cDNA was synthesized by reverse transcription of RNA pooled from representative developmental stages covering the entire larval and early post-metamorphic development (cDNA synthesis kit, #04379012001, Roche Diagnostics). Hox gene sequences were amplified with the gene-specific primers via standard PCR. PCR products were cloned by insertion into pGEM-T easy vectors (#A1360, Promega) and plasmid minipreps were purified with Qia-miniprep kit (#27106, Qiagen). Antisense and sense probes from linearized plasmid sequences were synthesized with a DIG-labeling kit (#11277073910, Roche Diagnostics).