Figure 2

Antibody specificity; (A) Igf-2 immunostaining of adjacent sections of an e13.5 embryo illustrate positive Igf-2 staining (brown) in liver (liv) and diaphragm (dia) which, (B) is completely abolished by pre-incubation of the antibody with 2ug/ml Igf-2 (pep; peptide absorption) demonstrating the specificity of Igf-2 staining. (C-D) Immunoblotting of whole embryo protein (E12.5 – E17.5) with, (C) clone My32 anti-FMyHC antibody: detects three positive bands corresponding to the three isoforms of adult fast myosin heavy chain (FMyHC) 2b, 2a and 2x/d. E12.5 and E13.5 tracks contain 20 μg protein. E15.5 and E17.5 tracks are loaded with 10 μg protein to prevent over-exposure of FMyHC, this is reflected in the intensity of bands in the anti-Spectrin loading control. (D) Because My32 is reported to cross-react with neonatal MyHC in rat we compared My32 staining with an anti-neonatal MyHC antibody (clone WB-MHCn) which detects a weak band corresponding to neonatal MyHC and coincident with FMyHC 2b. We conclude that the predominant FMyHC forms detected by My32 in mouse embryos are therefore Adult FMyHC 2a, 2x and 2b. WB-MHCn weakly cross-reacts with the adult fast myosin marker (track M).