Albino embryos are preferred, because pigmentation in wild-type embryos can obscure the hybridization signal. If pigmented embryos or explants are to be used, they can be taken through the procedure and then bleached afterwards by incubating in 10% hydrogen peroxide in methanol. Satisfactory results can be obtained in this manner. The bleaching can take from several hours to several days depending on the pigmentation of the embryos.
Just before fixation, embryos should be removed from their vitelline membranes using sharpened watchmaker's forceps. For blastula and gastrula stage embryos, a hole should be placed in the blastocoel to prevent nonspecific trapping of the probe and/or antibody. For neurula stages, the archenteron should be pierced. Embryos are fixed in small glass vials or scintillation vials if a large number are to be processed simultaneously. The preferred fixative is MEMFA (0.1 M MOPS, pH 7.4, 2 mM EGTA, 1 mM MgSO4, 3.7% formaldehyde). A 10X stock solution of the salts can be kept at 4°C. To make fresh fixative, add 1/10 vol 37% formaldehyde and 1/10 vol 10X salts and water to make the volume needed. Embryos should be fixed for approx 1 h at room temperature with constant rotation. After fixation, remove the fixative, and add 100% ethanol or methanol to the embryos. Make two changes of the 100% alcohol, and store the embryos at -20°C. Embryos can be stored for several months or longer.
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