Antibody:	Anti-ZO-2
Assay notes:	The fixative was 0.5% paraformaldehyde.

Structure	Level	Pattern	Image	Note
TS28: secondary oocyte	Present	Diffuse	2A	The protein was diffusely distributed in the cytoplasm of unfertilized eggs particularly close to the metaphase spindle.

Structure	Level	Pattern	Image	Note
TS1: 1-cell stage embryo	Present	Regionally restricted	2B	Staining was seen as fine specks in and around both pronuclei.

Structure	Level	Pattern	Image	Note
TS1: 1-cell stage embryo	Present	Regionally restricted	2C	As the pronuclei began to move to the center of the zygote, staining became more prominent within the cytoplasm as well as within the pronuclei.

Structure	Level	Pattern	Image	Note
TS1: 1-cell stage embryo	Present	Regionally restricted	2D	As the pronuclei began to move to the center of the zygote, staining became more prominent within the cytoplasm as well as within the pronuclei.

Structure	Level	Pattern	Image	Note
TS2: 2-cell stage embryo	Present	Regionally restricted	2E	After the first cell division, most immunostaining specks were located in the cytoplasm close to the plane of cleavage.

Structure	Level	Pattern	Image	Note
TS2: 2-cell stage embryo	Present	Regionally restricted	2F	In late 2-cell embryos, most immunostaining was present within the nuclei of the two blastomeres but excluded from the nucleoli.

Structure	Level	Pattern	Image	Note
TS3: 4-cell stage embryo	Present	Regionally restricted	3A	Expression was localized within all nuclei but was excluded from the nucleoli.

Structure	Level	Pattern	Image	Note
TS3: 8-cell stage embryo	Present	Regionally restricted	3B	Expression was localized within all nuclei but was excluded from the nucleoli.

Structure	Level	Pattern	Image	Note
TS3: 8-cell stage embryo	Present	Regionally restricted	3C	Expression was localized within all nuclei but was excluded from the nucleoli.

Structure	Level	Pattern	Image	Note
TS3: 8-cell stage embryo	Present	Regionally restricted	3D	Following compaction, nuclear expression diminished in intensity with gradually more nuclei becoming unlabeled in late cleavage.

Structure	Level	Pattern	Image	Note
TS3: compacted morula	Present	Regionally restricted	3E	Nuclear expression diminished in intensity with gradually more nuclei becoming unlabeled in late cleavage. Distinct membrane staining was first detected at the 16-cell stage as a discontinuous linear array of spots at cell boundaries between outer (trophectoderm lineage) blastomeres.

Structure	Level	Pattern	Image	Note
TS4: compacted morula	Present	Regionally restricted	3F	Nuclear expression diminished in intensity with gradually more nuclei becoming unlabeled in late cleavage. At the late morula stage, immunostaining changed to a continuous, linear pattern around blastomeres. Mid-plane section of late morulae was seen as punctuate dots at the apicolateral membrane regions of outer cells.

Structure	Level	Pattern	Image	Note
TS4: blastocyst	Present	Regionally restricted	3G	Nuclear expression diminished in intensity with gradually more nuclei becoming unlabeled in late cleavage. The continuous linear staining pattern increased in early blastocyst stage. Mid-plane section of early blastocysts was seen as punctuate dots at the apicolateral membrane regions of outer cells.
TS4: inner cell mass	Absent		3G

Structure	Level	Pattern	Image	Note
TS4: blastocyst	Present	Regionally restricted	3H	Nuclear expression diminished in intensity with gradually more nuclei becoming unlabeled in late cleavage. The continuous linear staining pattern increased in early blastocyst stage. In tangential sections, expression encircled neighboring cells of the embryo.

Structure	Level	Pattern	Image	Note
TS5: blastocyst	Present	Regionally restricted	3I	Nuclear expression diminished in intensity with gradually more nuclei becoming unlabeled in late cleavage. The continuous linear staining pattern increased in late blastocyst stage. Mid-plane section of blastocysts was seen as punctuate dots at the apicolateral membrane regions of outer cells.
TS5: inner cell mass	Absent		3I

Structure	Level	Pattern	Image	Note
TS5: blastocyst	Present	Regionally restricted	3J	Nuclear expression diminished in intensity with gradually more nuclei becoming unlabeled in late cleavage. The continuous linear staining pattern increased in blastocyst stage. In tangential sections, expression encircled neighboring cells of the embryo.

Structure	Level	Pattern	Image	Note
TS3: compacted morula	Present	Regionally restricted	4A Merged , 4A ZO-2	Double staining revealed co-localization of Tjp2 and Cdh1 in the apicolateral junctional complex.

Structure	Level	Pattern	Image	Note
TS4: blastocyst	Present	Regionally restricted	4B Merged , 4B ZO-2	In early blastocysts Tjp2 is apical to and distinct from Cdh1, which was localized in the apicolateral junctional complex.

Structure	Level	Pattern	Image	Note
TS4: blastocyst	Present	Regionally restricted	4C Merged , 4C ZO-2	Double staining with Tjp2 and Ocln showed co-localization of staining in the apicolateral tight junctions of the outer trophectoderm cells.