Antibody:	Anti-smooth muscle alpha-Actin (clone 1A4)
Detection system:	Secondary antibody coupled to horseradish peroxidase
Assay notes:	Amsterdam's fixative was used: methanol:acetone:acetic acid:water = 35:35:5:25 v/v.

Structure	Level	Pattern	Image	Note
TS16: outflow tract mesenchyme	Present	Regionally restricted	1B	Expression was detected in mesenchymal cells migrating into the endocardial ridges of the outflow tract.

Structure	Level	Pattern	Image	Note
TS13: aortic sac	Absent		2A	The roof of the aortic sac was negative for staining.
TS13: cardiac muscle tissue	Present	Uniform	2A
TS13: outflow tract endocardial tube	Absent		2A	No positive cells were found in either in the endocardial outflow tract ridges or in the atrioventricular cushions.

Structure	Level	Pattern	Image	Note
TS15: outflow tract	Present	Regionally restricted	2B , 2E	Expression was detected in the anlage of the aortico-pulmonary septum (APS). In the region of the future APS, underneath the immunopositive cells, an area of condensed mesenchyme is negative for staining.
TS15: outflow tract endocardial cushion	Present	Regionally restricted	2B , 2E	Expression was detected in the endocardial ridges.
TS15: cardiac muscle tissue	Present	Uniform	2B

Structure	Level	Pattern	Image	Note
TS16: arterial system	Present	Regionally restricted	2C	The aortic arch arteries and their derivatives were immunopositive.
TS16: outflow tract mesenchyme	Present		2C , 2F
TS16: outflow tract endocardial cushion	Present	Regionally restricted	2C , 2F	Cohorts of immunopositive cells penetrate deeper (i.e. more proximal) into the endocardial ridges.
TS16: aortico-pulmonary spiral septum	Strong	Regionally restricted	2C , 2F	The leading edge of the aortico-pulmonary septum was heavily populated with immunopositive cells. However, the condensed mesenchymal core was immunonegative.
TS16: cardiac muscle tissue	Present	Uniform	2C

Structure	Level	Pattern	Image	Note
TS18: dorsal aorta	Present	Regionally restricted	2D	Expression was detected in the smooth muscle cells.
TS18: arterial system	Present	Regionally restricted	2D	The aortic arch arteries and their derivatives were immunopositive.
TS18: outflow tract endocardial cushion	Present	Regionally restricted	2G , 2D	Cohorts of immunopositive cells penetrate deeper (i.e. more proximal) into the endocardial ridges.
TS18: aortico-pulmonary spiral septum	Strong	Regionally restricted	2G , 2D	The leading edge of the aortico-pulmonary septum was heavily populated with immunopositive cells. However, the condensed mesenchymal core was immunonegative.
TS18: outflow tract	Present	Regionally restricted	2G , 2D	Immunopositive cells were detected in the outflow tract mesenchyme.
TS18: cardiac muscle tissue	Present	Uniform	2D

Structure	Level	Pattern	Image	Note
TS19: aortico-pulmonary spiral septum	Present		3A
TS19: outflow tract endocardial cushion	Present		3A	Expression was detected in the outflow tract ridges.

Structure	Level	Pattern	Image	Note
TS20: aortico-pulmonary spiral septum	Present		3C , 3D
TS20: outflow tract endocardial cushion	Present		3C , 3D

Structure	Level	Pattern	Image	Note
TS21: heart septum	Strong	Regionally restricted	4A , 4C	A heavy band of immunopositive cells were detected in the developing conal septum.
TS21: cardiac muscle tissue	Present		4A , 4C

Structure	Level	Pattern	Image	Note
TS21: heart mesenchyme	Present	Regionally restricted	4D	Expression was detected in a band of immunopositive mesenchymal cells between the aortic and pulmonary valves in the conal septum.
TS21: heart septum	Present	Regionally restricted	4D	Expression was detected in a band of immunopositive mesenchymal cells between the aortic and pulmonary valves in the conal septum.

Structure	Level	Pattern	Image	Note
TS22: heart mesenchyme	Present	Regionally restricted	5A	Expression was detected in mesenchymal cells in the conal septum. However, there were immunonegative condensed mesenchymal cells in the conal septum.
TS22: heart septum	Present	Regionally restricted	5A	Expression was detected in mesenchymal cells in the conal septum. However, there were immunonegative condensed mesenchymal cells in the conal septum.

Structure	Level	Pattern	Image	Note
TS16: outflow tract mesenchyme	Absent		6A	There was no immunostaining in connective tissue, outflow tract ridges, or in the region of the aortico-pulmonary septum.
TS16: cardiac muscle tissue	Present		6A

Structure	Level	Pattern	Image	Note
TS18: aortic sac	Present		6B

Structure	Level	Pattern	Image	Note
TS18: outflow tract	Present	Regionally restricted	6C	Expression was detected in the distal outflow tract ridges. More immunopositive cells were noted on the rightward aspect.

Structure	Level	Pattern	Image	Note
TS19: outflow tract endocardial cushion	Present	Regionally restricted	6D	Expression was detected in the distal cushions. More immunopositive cells were noted on the rightward aspect.

Structure	Level	Pattern	Image	Note
TS20: outflow tract	Strong	Regionally restricted	7A	Expression was strong in a midline position in the very distal portion of the outflow tract ridges.

Structure	Level	Pattern	Image	Note
TS20: outflow tract	Weak	Regionally restricted	7B	Faint staining (if any) was noted in the proximal outflow tract ridges.

Structure	Level	Pattern	Image	Note
TS20: outflow tract	Present	Regionally restricted	7C	Expression was detected in mesenchymal cells in the distal area of the outflow tract.

Structure	Level	Pattern	Image	Note
TS20: heart	Present		8A

Structure	Level	Pattern	Image	Note
TS20: outflow tract	Present	Regionally restricted	8B	Expression was detected within the outflow tract ridges to the left on the ventral aspect of the outflow tract.

Structure	Level	Pattern	Image	Note
TS21: heart mesenchyme	Present	Regionally restricted	8D	Expression was detected in under-developed dorsal and ventral prongs of condensed mesenchyme above the level of the abnormally developing truncal valve.

Structure	Level	Pattern	Image	Note
TS22: heart mesenchyme	Present	Regionally restricted	8E	Expression was detected in a large group of mesenchymal cells; within that group there was a cluster of immunonegative condensed mesenchyme.

Structure	Level	Pattern	Image	Note
TS22: heart mesenchyme	Present	Regionally restricted	8F	A ridge, possible representing the remnant of the dorsal prong of immunopositive mesenchyme, was seen near the truncal valve level.

Structure	Level	Pattern	Image	Note
TS22: aortico-pulmonary spiral septum	Strong		9B , 9C

Structure	Level	Pattern	Image	Note
TS22: heart	Present		9E , 9F