Bm purple substrate

After micro-CT scanning, the tibiae were decalcified in 19%
ethylenediaminetetraacetic acid (EDTA) for approximately 3 weeks at 4
°C. Decalcified samples were dehydrated in a graded ethanol series,
embedded into paraffin, and cut into 10-μm thick sections. Aniline blue
staining, which labels osteoid matrix, was used to detect osseous tissues as
previously described (Kim et al., 2007 (link)).
Histochemistry for alkaline phosphatase (ALP) and tartrate-resistant acid
phosphatase (TRAP) were performed with BM-purple substrate (Roche, Indianapolis,
IN) and Leucocyte Acid Phosphatase kit (Sigma, St. Louis, MO), respectively,
according to manufacturers’ instructions. Proliferating cell nuclear
antigen (PCNA) immunohistochemistry was performed to assess cell proliferation
at the injury site by using the anti-PCNA antibody (PCNA D3H8O XP rabbit, Cell
Signaling Technologies, Danvers, MA) which was detected by
streptavidin-conjugated horseradish peroxidase (HRP) and diaminobenzidine (DAB).
followed by 4′,6-diamidino-2-phenylindole (DAPI) nuclear stain (Sigma).
DAPI was used to count the total number of cells in the region of interest to
serve as the denominator for the quantification of PCNA-positive cells. The
stained sections were examined and photographed using a Leica digital imaging
system.

An Msx1 1.2kb XhoI-XbaI cDNA fragment was subcloned into pSP72. Msx2 full-length cDNA was cloned into pBSKII(+) between BamHI and EcoRI sites. The Blimp1 cDNA construct is a kind gift from Dr. Saitou at RIKEN Kobe Institute, Japan (Ohinata et al., 2005 (link)). The Fragilis cDNA probe was obtained from Dr. Surani at Wellcome Trust/Cancer Research UK Institute (Saitou et al., 2002 (link)). The Wnt5a probe was generously provided by Dr. SK Dey at Cincinnati Children’s Hospital Medical Center (Daikoku et al., 2011 (link)). A 700bp Fibronectin cDNA probe was generated by PCR with forward 5′-AGA TGA CTC ATG CTT TGA CCC-3′ and reverse 5′-TGC TGA AGC TGA GAA CAT GGC-3′ primers. Ribonucleotide probes were synthesized by PCR with either Fluorescein-UTP or Digoxygenin-UTP (Roche Applied Science). Whole-mount in situ hybridization was performed largely based on the method reported by Hogan and visualized by BM-purple substrate (Roche Applied Science) (Hogan et al., 1994 ). Prior to sectioning, embryos were fixed overnight in 4% PFA/PBS and embedded in HistoPrep (Fisher). In situ hybridization on frozen sections was performed as previously described and fluorophore-conjugated Tyramide (TSA™PLUS, Perkin Elmer) was used to amplify the signal (Ting et al., 2009 (link)).