Embryo Tissue Analysis Protocol

Limbs and lumbar vertebrae dissected from sacrificed embryos were fixed in 4% PFA for 24 h at 4°C, decalcified in 10% (w/v) EDTA /7% (v/v) glycerol (pH 7.4) for 48 h at 4°C, and embedded in paraffin. Hematoxylin and eosin staining was performed using 6-μm paraffin sections according to standard protocols. Apoptotic cells were detected using TUNEL staining with an In Situ Apoptosis Detection Kit (TaKaRa). The frequencies of BrdU- and TUNEL-positive cells were calculated using ImageJ software. In situ hybridization was performed by Genostaff Co., Ltd (Tokyo, Japan) as described previously [35 (link)]. For the analysis of cell proliferation, we injected pregnant mice intraperitoneally with 100 μg of BrdU/g of body weight 1 h before sacrifice. BrdU-labeled cells incorporated on the sections from BrdU-treated embryos were stained with a BrdU Immunohistochemistry Kit (Abcam, Cambridge, MA, UK).