Immunostaining of Embryonic Proteins

Intact embryos were fixed in 2.5%PFA/ethanol [95 (link)] or methanol/acetone [96 (link)] for all immunofluorescence except for those probed with monoclonal antibody H5, which was fixed in methanol/formaldehyde [95 (link)]. Primary antibodies used were: anti-H3K4me2 (CMA30) 1:1000 END Millipore], anti-P-granules [OIC1D4, 1:5 [96 (link), 97 (link)])], anti-H3K36me3 [(CMA333), 1:1000 [95 (link)]], anti-Ser2p RNA pol II CTD (H5, 1:500, Covance MMS-129R), anti-GFP (1:1000, Novus NB600-308), anti-AMA-1 (1:10,000, Novus 38520002), and anti-FLAG (M2, 1:1000, Sigma F1804). Secondary antibodies used were Alexa Fluor 488-conjugated donkey anti-mouse (1:500, Invitrogen R37114) and Alexa Fluor 594-conjugated goat anti-rabbit (1:500, Invitrogen R37117). Samples were mounted in ProLong Gold anti-fade reagent (Life technologies, P36934) and observed under a fluorescence microscope (Leica DMRXA; Hamamatsu Photonics, Hamamatsu, Japan) with Simple PCI software (Hamamatsu Photonics). Image J was used for quantification of raw immunofluorescence intensity [98 ].