M250c stereomicroscope

In situ hybridization of whole embryonic limbs to detect Gdf5 and Sox9 was carried out as described (Wilkinson 1992 ). An acetylation step prior to hybridization was necessary for Gdf5, but not for Sox9. Plasmid containing Sox9 (cDNA clone of nt, 116–856; NM_011448) or Gdf5 (cDNA clone of bp 1321–1871; NM_008109), generously provided by Dr. Eiki Koyama, was transcribed into anti-sense and sense probes using digoxygenin-labeled dNTPs (Fisher). Hybridized samples were incubated with a 1:2000 dilution of alkaline phosphatase-conjugated anti-DIG antibody (Cell Signaling) and developed using an alkaline phosphatase chromogenic substrate (BM Purple, Sigma-Aldrich).
Whole mount in situ hybridization using synthesized digoxygenin labeled LNA probe for murine Acvr1 (Exiqon) followed the recommended Exiqon protocol (Sweetman et al 2006 (link), 2008 (link)). Digoxygenin was detected using Vector Blue Alkaline Phosphatase Substrate Kit (SK-5300, Vector Laboratories).
Samples were imaged using an M250C Leica stereomicroscope fitted with a Leica DFC450 C camera.

Skeletal tissue dissection, processing, and staining were performed by standard methods (McLeod 1980). Skeletal elements were stained with Alizarin Red S (mineralized tissue/bone; Sigma) and Alcian Blue GS (glycose-amino-glycans/cartilage; Sigma), then cleared and preserved in glycerol for imaging and analysis using an M250C Leica stereomicroscope fitted with a Leica DFC450 C camera.