Identification of a second follistatin gene in grass carp (Ctenopharyngodon idellus) and its regulatory function in myogenesis during embryogenesis

Duplicated connective tissue growth factor genes in hypoxia-sensitive blunt snout bream Megalobrama amblycephala and their in vivo expression

“…Embryos were collected for in situ hybridization, manually dechorionated, and fixed overnight at 4°C in 4% phosphate-buffered paraformaldehyde. Fixed blunt snout bream embryos were washed briefly in phosphate-buffered saline (PBS) containing 0.1% Tween-20, transferred to 100% methanol and stored at −20°C for a minimum of 24 h. Whole-mount in situ hybridization using digoxigenin (DIG)-labeled RNA riboprobes was carried out essentially as reported previously, but with certain modifications (Jiang et al, 2012;Zhong et al, 2013). Briefly, embryos were hybridized with appropriate riboprobes at 60°C, incubated with antiDIG antibodies conjugated with alkaline phosphatase (AP), and stained with Roche BM Purple AP substrates (Roche, Basel, Switzerland) to produce purple, insoluble precipitates.…”