resource source identifier antibodies t bra brachyury n 19  (Santa Cruz Biotechnology)

Journal: bioRxiv

Article Title: The emergence of human gastrulation upon in vitro attachment

doi: 10.1101/2023.05.16.541017

Figure Lengend Snippet: A) Brightfield image of PALLY-LY differentiated blastoids in Aggrewells. B) Immunostaining blastoids displays OCT4 (green), SOX17 (magenta), and GATA3 (blue) marker genes distinguishing epiblast, primitive endoderm, and trophectoderm. Scale bar 100um. C) In vitro attached human blastoids analyzed by immunostaining show OCT4 + /BRA + cells 7 dpa. OCT4 + cells label the epiblast territory, GATA6 + the primitive endoderm, and BRA + the primitive streak and mesoderm populations. Immunostaining pseudo colors: OCT4 (green), BRA (red), GATA6 (magenta), and merge with DAPI (grey). Scale bar: 100um. D) Immunostaining at 10 dpa displays OCT4 + -epiblast, BRA + -primitive streak and mesoderm, and GATA6 + -endoderm cells. Immunostaining pseudo colors: OCT4 (green), BRA (red), GATA6 (magenta), and merge with DAPI (grey). Scale bar: 100um. E) Scaled area quantification of OCT4 + and BRA + cells at 7 and 10 dpa. Data are displayed as a boxplot (center line displays the median, and box limits are the 25th and 75th percentiles) (n = 51 from 3 independent differentiations). Student’s t-test, unpaired with two tails. ***p<0.001. F) Pie chart of in vitro attached blastoids that broke symmetry at 7 and 10 dpa. G) Area quantification is displayed as a scatterplot. x-axis reports the scaled OCT4 while the y-axis is the BRA scaled area. Each dot represents an individual attached blastoid that has been color-coded according to the time of analysis. Grey dots represent blastoids 7 dpa and blue dots blastoids at 10 dpa.

Article Snippet: List of primary antibodies: OCT4 Santa Cruz Biotechnology (sc-5279) 1:200 BRA R&D Systems (MAB20851-100) 1:500 GATA6 R&D Systems (AF1700) 1:200 GATA3 Thermo Fisher Scientific (14-9966-82) 1:100 SOX17 R&D Systems (MAB1924) 1:200 HAND1 R&D Systems (AF3168-SP) 1:200 KRT7 Abcam (ab209600) 1:100 ISL1 DSHB (39.4D5) 1:200 MIXL1 Sigma Prestige antibody (HPA005662) 1:200 SUSD2 Miltenyi Biotec (130-106-401) 1:100 SSEA4 Miltenyi Biotec (130-122-958) 1:100 KLF17 Atlas Antibodies (HPA024629) 1:200 SOX17 R&D Systems (AF2864) 1:200 GATA4 Thermo Fisher Scientific (14-9980-82) 1:100 FOXA2 Novus Biologicals (AF2400) 1:200

Techniques: Immunostaining, Marker, In Vitro

Journal: bioRxiv

Article Title: The emergence of human gastrulation upon in vitro attachment

doi: 10.1101/2023.05.16.541017

Figure Lengend Snippet: Primitive streak and mesoderm induction upon in vitro attachment of human blastoids A) Immunostaining of in vitro attached blastoids derived from at 10 dpa sorted by the size of the OCT4 positive epiblast (small, medium, and large) and whether BRA is induced (red bar = broken symmetry and grey bar = unbroken symmetry). Immunostaining pseudo colors: OCT4 (green), BRA (red), and DAPI (grey). Scale bar: 100um. B) Immunostaining of in vitro attached blastoids at 10 dpa derived from an additional pluripotent stem cell line (niPSC75.2, ). Immunostaining pseudo colors: OCT4 (green), BRA (red), GATA6 (magenta) and DAPI (grey). Scale bar: 100um.

Article Snippet: List of primary antibodies: OCT4 Santa Cruz Biotechnology (sc-5279) 1:200 BRA R&D Systems (MAB20851-100) 1:500 GATA6 R&D Systems (AF1700) 1:200 GATA3 Thermo Fisher Scientific (14-9966-82) 1:100 SOX17 R&D Systems (MAB1924) 1:200 HAND1 R&D Systems (AF3168-SP) 1:200 KRT7 Abcam (ab209600) 1:100 ISL1 DSHB (39.4D5) 1:200 MIXL1 Sigma Prestige antibody (HPA005662) 1:200 SUSD2 Miltenyi Biotec (130-106-401) 1:100 SSEA4 Miltenyi Biotec (130-122-958) 1:100 KLF17 Atlas Antibodies (HPA024629) 1:200 SOX17 R&D Systems (AF2864) 1:200 GATA4 Thermo Fisher Scientific (14-9980-82) 1:100 FOXA2 Novus Biologicals (AF2400) 1:200

Techniques: In Vitro, Immunostaining, Derivative Assay

Journal: bioRxiv

Article Title: The emergence of human gastrulation upon in vitro attachment

doi: 10.1101/2023.05.16.541017

Figure Lengend Snippet: Extra-embryonic cells of the trophectoderm and the amnion at 10 dpa. A) Immunostaining of in vitro attached blastoids displayed surrounding GATA3/KRT7 positive cells at 10 dpa. The epiblast population is identified using OCT4 (green), the primitive streak/mesoderm population using BRA (red), GATA3 and KRT7 extraembryonic ectoderm population using GATA3/KRT7 (blue and magenta). Scale bar: 100um. B) Immunostaining of in vitro attached blastoids at 10 dpa. The epiblast population is identified using SOX2 (cyan), the primitive streak/mesoderm population using BRA (red), the amnion population using ISL1 (green). Scale bar: 100um.

Article Snippet: List of primary antibodies: OCT4 Santa Cruz Biotechnology (sc-5279) 1:200 BRA R&D Systems (MAB20851-100) 1:500 GATA6 R&D Systems (AF1700) 1:200 GATA3 Thermo Fisher Scientific (14-9966-82) 1:100 SOX17 R&D Systems (MAB1924) 1:200 HAND1 R&D Systems (AF3168-SP) 1:200 KRT7 Abcam (ab209600) 1:100 ISL1 DSHB (39.4D5) 1:200 MIXL1 Sigma Prestige antibody (HPA005662) 1:200 SUSD2 Miltenyi Biotec (130-106-401) 1:100 SSEA4 Miltenyi Biotec (130-122-958) 1:100 KLF17 Atlas Antibodies (HPA024629) 1:200 SOX17 R&D Systems (AF2864) 1:200 GATA4 Thermo Fisher Scientific (14-9980-82) 1:100 FOXA2 Novus Biologicals (AF2400) 1:200

Techniques: Immunostaining, In Vitro

Journal: bioRxiv

Article Title: The emergence of human gastrulation upon in vitro attachment

doi: 10.1101/2023.05.16.541017

Figure Lengend Snippet: A) Immunostaining of the in vitro attached human embryo at 12 dpf. A low-magnification field of view shows DAPI (cyan), GATA6 (magenta), OCT4 (green) and BRA (red) signals, all overlayed on the F-ACTIN (grey) staining. OCT4 localizes the epiblast territory and GATA6 the primitive endoderm cells. BRA is used as a primitive streak and mesoderm marker. Scale bar: 100um. B) Immunostaining of the in vitro attached human embryo at 12 dpf. Zoomed-in field of view shows the merged image of OCT4 (green), BRA (red), GATA6 (magenta) and F-ACTIN (grey) signals. Scale bar: 50um. C) 3D orthogonal projections of the in vitro attached human embryo at 12 dpf. Top and lateral views show the merge of OCT4 (green), BRA (red), GATA6 (magenta), and DAPI (grey). Scale bar: 50um.

Article Snippet: List of primary antibodies: OCT4 Santa Cruz Biotechnology (sc-5279) 1:200 BRA R&D Systems (MAB20851-100) 1:500 GATA6 R&D Systems (AF1700) 1:200 GATA3 Thermo Fisher Scientific (14-9966-82) 1:100 SOX17 R&D Systems (MAB1924) 1:200 HAND1 R&D Systems (AF3168-SP) 1:200 KRT7 Abcam (ab209600) 1:100 ISL1 DSHB (39.4D5) 1:200 MIXL1 Sigma Prestige antibody (HPA005662) 1:200 SUSD2 Miltenyi Biotec (130-106-401) 1:100 SSEA4 Miltenyi Biotec (130-122-958) 1:100 KLF17 Atlas Antibodies (HPA024629) 1:200 SOX17 R&D Systems (AF2864) 1:200 GATA4 Thermo Fisher Scientific (14-9980-82) 1:100 FOXA2 Novus Biologicals (AF2400) 1:200

Techniques: Immunostaining, In Vitro, Staining, Marker

Journal: bioRxiv

Article Title: The emergence of human gastrulation upon in vitro attachment

doi: 10.1101/2023.05.16.541017

Figure Lengend Snippet: In vitro attached human embryos break epiblast symmetry and self-organize a BRA-positive population at 12 dpf in the absence of Laminin-521 coated plastic substrates. C) Immunostaining of in vitro attached human embryos at 12 dpf. Large field and zoomed-in views show DAPI (cyan), F-ACTIN (grey), OCT4 (green) and BRA (red) signals. Scale bar: 100um.

Article Snippet: List of primary antibodies: OCT4 Santa Cruz Biotechnology (sc-5279) 1:200 BRA R&D Systems (MAB20851-100) 1:500 GATA6 R&D Systems (AF1700) 1:200 GATA3 Thermo Fisher Scientific (14-9966-82) 1:100 SOX17 R&D Systems (MAB1924) 1:200 HAND1 R&D Systems (AF3168-SP) 1:200 KRT7 Abcam (ab209600) 1:100 ISL1 DSHB (39.4D5) 1:200 MIXL1 Sigma Prestige antibody (HPA005662) 1:200 SUSD2 Miltenyi Biotec (130-106-401) 1:100 SSEA4 Miltenyi Biotec (130-122-958) 1:100 KLF17 Atlas Antibodies (HPA024629) 1:200 SOX17 R&D Systems (AF2864) 1:200 GATA4 Thermo Fisher Scientific (14-9980-82) 1:100 FOXA2 Novus Biologicals (AF2400) 1:200

Techniques: In Vitro, Immunostaining