Table 1.

Summary of early PTA-staining protocols.

Reference	Ethanol dehydration	PTA Staining	Notes
(Metscher, 2009a)	“Take samples to 70% ethanol.”	0.3% PTA in 70% ethanol “Stain overnight or longer.”	“The staining times were found not to be critical, as long as the stain had sufficient time to penetrate the tissues.”
(Metscher, 2009b)	“Samples in 70% ethanol.”	0.3% PTA in 70% ethanol “Stain 2 hr to overnight.”	“Slow tissue penetration, limited to a few millimeters.”
(Metscher, 2011)	“Rinse in water. Transfer to 35% ethanol and leave for 10–15 min. Transfer to 70% ethanol and leave for 10–15 min or longer.”	0.3% PTA in 70% ethanol “Stain in PTA for 2 h to overnight.”	“The staining time is not critical and PTA does not overstain, but larger embryos (e.g., E12 or later mice) should be stained overnight. PTA has trouble penetrating more than about 2 mm of tissue.” “The PTA concentration is not critical, nor is the grade and concentration of alcohol.”