The protocol of ISH was previously described (16 (link)). Specific regions of lefty1 and foxj1a were cloned into pGEM T easy vector (Promega). ISH probe vectors for cmlc1, dand5, spaw, and pan-akap12 were previously described (12 (link), 16 (link)) and primer sequences of lefty1 and foxj1a for ISH probe vectors are summarized in Supplementary Table S1 . The protocol of whole-mount immunostaining for Tg(sox17:egfp) embryos was described previously (16 (link)). Mouse anti-Cdh1 (1:200, BD Biosciences) and goat anti-mouse AF546 (1:1000, Invitrogen) were used for immunofluorescence. Stained embryos were mounted in glycerol and images were obtained by an AxioCam ICC-1 camera (Zeiss) on a Stemi 2000C (Zeiss) for immunohistochemistry and an LSM700 confocal microscope (Zeiss) for immunofluorescence, respectively, and processed using ZEN 2012 software (Zeiss).
Protocol detail
In Situ Hybridization and Immunostaining Protocol
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pGEM T easy vector
Mouse anti-Cdh1
goat anti-mouse AF546
AxioCam ICC-1 camera
Stemi 2000C
LSM700 confocal microscope
ZEN 2012 software
Akap12
Cdh1
Confocal microscope
Embryos
Glycerol
Goat
Immunofluorescence
Immunohistochemistry
Lefty1
Mouse
Pgem
Primer
Promega
Stemi
Vectors
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Variable analysis
independent variables
- Specific regions of lefty1 and foxj1a were cloned into pGEM T easy vector (Promega)
- Expression patterns of lefty1, foxj1a, cmlc1, dand5, spaw, and pan-akap12
- The protocol of ISH was previously described (16)
- The protocol of whole-mount immunostaining for Tg(sox17:egfp) embryos was described previously (16)
- ISH probe vectors for cmlc1, dand5, spaw, and pan-akap12 were previously described (12, 16)
- No negative controls explicitly mentioned
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