vcam-1/cd106 antibody (6g9) - bsa free  (Bio-Techne corporation)

Journal: Nature Communications

Article Title: A microphysiological model of human trophoblast invasion during implantation

doi: 10.1038/s41467-022-28663-4

Figure Lengend Snippet: a Four different culture conditions investigated in proteomics analysis. b Plots generated by principal component analysis performed across all quantifiable proteins ( n = 498) from 4 biological replicates for each of the tested conditions. Each data point represents an individual biological replicate. c Heirarchical clustering plot. d Heatmap depicting the human proteins significantly modulated in abundance in the different tested conditions (one-way ANOVA p < 0.05). e , f 19 and 9 proteins without known function in implantation from the comparison of secretome e between CO and EVT-mono and f between CO and EC-mono, respectively. g 17 uniquely expressed endothelial proteins in the coculture configuration of the implantation-on-a-chip, including 8 proteins with unknown function in implantation and placental development. The left-most column named Ref. shows references in Supplementary Information. Immunofluorescence of h VCAM-1 and i C4 in the vascular endothelium in the implantation-on-a-chip at Day 6. The representative images are from three independent experiments. Scale bars, 50 μm. j Quantification of immunofluorescence of VCAM-1 and C4 staining. Data were presented as mean ± s.d. Two-sided t-test ( n = 8 images taken from four independent devices per group). p values are shown on graphs. k 18 uniquely induced EVT proteins in coculture, including 8 proteins with previously unknown roles in implantation/placentation. The left-most column named Ref. shows references in Supplementary Information. Source data are provided as a Source Data file.

Article Snippet: Cells were fixed by introducing 4% paraformaldehyde (PFA, Thermo Scientific, catalog # AAJ19943K2) into the channels and incubated for 30 min at room temperature (RT), followed by 3 washes with PBS for all immunofluorescence experiments with the exception that HLA-G MEM-G/9 staining was carried out after cold acetone fixation at −20 degrees for 10 min. After cell permeabilization and blocking with 0.1% Triton-X and 1% bovine serum albumin (BSA, Sigma, catalog # 5217), respectively, the cells were incubated with primary antibodies against Cytokeratin-7 (EPR17078, Abcam, catalog # ab181598), CD-31 (P2B1, Abcam, catalog # ab24590), VE-cadherin (D87F2, Cell Signaling, catalog # 2500), Caspase-3 (Asp175, Cell Signaling, catalog # 9661), Prolactin (PRL02, Thermo Fisher, MA5-11998), Fibroblast surface protein (1B10, Abcam, catalog # ab11333), Ki67 (37C7-12, Abcam, catalog # ab245113) VCAM-1(6G9, Novus Biologicals, NBP1-47491), and Complement C4 (JM88-13, Thermo Fisher, catalog # MA5-32856) diluted at 1:200 in 1% BSA-containing PBS solution at 4 °C overnight.

Techniques: Generated, Comparison, Immunofluorescence, Staining

Journal: Nature Communications

Article Title: A microphysiological model of human trophoblast invasion during implantation

doi: 10.1038/s41467-022-28663-4

Figure Lengend Snippet: a Soon after implantation, EVTs begin to differentiate from precursor cells in the cytotrophoblast shell (CS) and invade into the uterus, a process that continues through the first half of pregnancy. b Compartmentalized design of the implantation-on-a-chip device for in vitro modeling of EVTs and a maternal SA separated by maternal endometrium. c Architecture of the implantation-on-a-chip microdevice. The center and two side lanes have dimensions of 0.5 mm (width) × 0.3 mm (height) and 0.25 mm (width) × 0.3 mm (height), respectively. d Sequential steps of model construction. e Time-lapse imaging of ECM hydrogel precursor (colored black) injection into the center lane of the device. f Images of device cross-section to show capillary pinning-based physical confinement of injected hydrogel solution (dark solution) in the center lane. Scale bar, 500 μm g (Top row) Photos of first trimester termination tissue and EVT outgrowth from the tissue explants. Scale bars, 1 mm (middle) and 200 μm (right). (Bottom row) The purity of the population was confirmed by immunostaining for cytokeratin 7, a trophoblast marker (magenta) and HLA-G, an EVT-specific marker (green). The representative images of the villous tissue are from five independent experiments. Scale bars, 200 μm. h Immunostaining of HLA-G (green) and Ki67 (magenta) expression by EVTs cultured on coverslips in a 6 well plate, after 3 passages. The representative images of EVTs are from four independent experiments. Scale bars, 50 μm. i Top-down confocal projection of the microengineered maternal-fetal interface at Day 1. EVTs in the fetal chamber were labeled with CellTracker Green (green). ECs were stained for VE-cadherin (magenta). The representative image is from three independent devices. Scale bar, 200 μm. j Endothelial tube in the vascular compartment at Day 1. Magenta and blue show VE-cadherin and nuclear staining, respectively. The representative images are from three independent devices. Scale bars, 100 μm (top) and 50 μm (bottom). EVTs: Extravillous trophoblasts, CS: Cytotrophoblastic shell, ECs: Endothelial cells, ECM : Extracellular matrix, CK7 : Cytokerain 7, HLA-G : human leukocyte antigen G.

Article Snippet: Cells were fixed by introducing 4% paraformaldehyde (PFA, Thermo Scientific, catalog # AAJ19943K2) into the channels and incubated for 30 min at room temperature (RT), followed by 3 washes with PBS for all immunofluorescence experiments with the exception that HLA-G MEM-G/9 staining was carried out after cold acetone fixation at −20 degrees for 10 min. After cell permeabilization and blocking with 0.1% Triton-X and 1% bovine serum albumin (BSA, Sigma, catalog # 5217), respectively, the cells were incubated with primary antibodies against Cytokeratin-7 (EPR17078, Abcam, catalog # ab181598), CD-31 (P2B1, Abcam, catalog # ab24590), VE-cadherin (D87F2, Cell Signaling, catalog # 2500), Caspase-3 (Asp175, Cell Signaling, catalog # 9661), Prolactin (PRL02, Thermo Fisher, MA5-11998), Fibroblast surface protein (1B10, Abcam, catalog # ab11333), Ki67 (37C7-12, Abcam, catalog # ab245113) VCAM-1(6G9, Novus Biologicals, NBP1-47491), and Complement C4 (JM88-13, Thermo Fisher, catalog # MA5-32856) diluted at 1:200 in 1% BSA-containing PBS solution at 4 °C overnight.

Techniques: In Vitro, Imaging, Injection, Immunostaining, Marker, Expressing, Cell Culture, Labeling, Staining

Journal: Nature Communications

Article Title: A microphysiological model of human trophoblast invasion during implantation

doi: 10.1038/s41467-022-28663-4

Figure Lengend Snippet: a Endothelial contribution to EVT invasion was examined by comparing monoculture of EVTs to coculture of EVTs and ECs. b Top-down images of EVT migration at Days 2, 4, and 6. EVTs in the fetal chamber (F) were stained with CellTracker Green and DAPI. ECs in the vascular chamber (V) were visualized by DAPI nuclear staining. Dashed lines indicate the boundary between the ECM hydrogel and the vascular compartment. The representative images are from three independent experiments. Scale bars, 200 μm. Quantification of c the number of invading cells, d invasion depth. Two-sided t-test ( n = 3 independent devices per group). The rose plots in e show the distribution of orientation angles between the major axis of invading cells and the horizontal line. The length of each spoke around the half-circle is proportional to the percentage of cells. The white arrow at the center indicates the direction pointing from the fetal to vascular chamber. (The plots indicate the orientation of 14 to 16 EVTs randomly selected from a representative device per group. The same trends were confirmed in 3 independent devices per group.) f Invasion of EVTs labeled with CellTracker green in coculture with different types of primary human ECs after 6 days of culture. Bottom panels show the endothelium with CD31 staining (red) in the vascular chamber. The representative images are from three independent experiments. Scale bars, 200 μm (top) and 50 μm (bottom). Invasive capacity was quantified by the number of invading cells and invasion depth at Day 6. One-way ANOVA with Tukey’s multiple comparison test ( n = 3 independent devices per group). g A microdevice containing a widening central ECM chamber was used to evaluate the effect of distance between the fetal and vascular chambers on EVT migration. h , i Imaging and quantification of EVT invasion in three separate regions at Day 6. The ranges of channel width are 500–650 μm (Region A), 675–825 μm (Region B), and 850–1000 μm (Region C). The representative image is from three independent experiments. Scale bar, 600 μm. One-way ANOVA with Tukey’s multiple comparison test ( n = 3 independent devices). EVT proliferation was evaluated by j , k immunostaining of Ki67 (magenta). The representative images are from three independent experiments. Scale bars in j , 200 μm. Two-way ANOVA with Sidak’s multiple comparison test ( n = 3 independent devices). l Comparison of MMP-2 and 9 production measured by ELISA. Two-sided t -test ( n = 4 independent devices per group). Data are presented as mean ± SD. p values are shown on graphs. n.s.; not significant. Source data are provided as a Source Data file.

Article Snippet: Cells were fixed by introducing 4% paraformaldehyde (PFA, Thermo Scientific, catalog # AAJ19943K2) into the channels and incubated for 30 min at room temperature (RT), followed by 3 washes with PBS for all immunofluorescence experiments with the exception that HLA-G MEM-G/9 staining was carried out after cold acetone fixation at −20 degrees for 10 min. After cell permeabilization and blocking with 0.1% Triton-X and 1% bovine serum albumin (BSA, Sigma, catalog # 5217), respectively, the cells were incubated with primary antibodies against Cytokeratin-7 (EPR17078, Abcam, catalog # ab181598), CD-31 (P2B1, Abcam, catalog # ab24590), VE-cadherin (D87F2, Cell Signaling, catalog # 2500), Caspase-3 (Asp175, Cell Signaling, catalog # 9661), Prolactin (PRL02, Thermo Fisher, MA5-11998), Fibroblast surface protein (1B10, Abcam, catalog # ab11333), Ki67 (37C7-12, Abcam, catalog # ab245113) VCAM-1(6G9, Novus Biologicals, NBP1-47491), and Complement C4 (JM88-13, Thermo Fisher, catalog # MA5-32856) diluted at 1:200 in 1% BSA-containing PBS solution at 4 °C overnight.

Techniques: Migration, Staining, Labeling, Comparison, Imaging, Immunostaining, Enzyme-linked Immunosorbent Assay