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counterstaining with dapi  (Dojindo Labs)


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    Dojindo Labs counterstaining with dapi
    Activation of Wnt/β-catenin signaling enhances STB differentiation. (A) A scheme of the culture conditions. Undifferentiated macTSCs were cultured with [CHIR(+)] or without [CHIR(−)] CHIR99021 for 3 days. Some cells were induced to differentiate in the medium containing dbcAMP [CHIR(−) cAMP and CHIR(+) cAMP] for another 6 days. (B) The relative expression level of a vCTB marker, TEAD4 . (C) Phase contrast images of cells on day 9. Multinucleated STB is outlined by a red line. Scale bar = 100 μm. (D, G) The relative expression levels of STB (D) and EVT (G) markers. For C, D, and G, the mean values (±SD) of technical triplicates of biological duplicates, normalized by the expression of ACTB , are shown relative to those in CHIR(−) (arbitrarily set as 1). Different letters represent significant differences ( p < 0.05). (E) Detection of multinucleated STBs by immunofluorescence for E-cadherin, CG, and <t>DAPI</t> staining. Scale bar = 100 μm. (F) The mean intensity ± SD (%) of STB nuclei calculated from four randomly chosen fields of biological duplicates. Different letters represent significant differences ( p < 0.05). Note that error bars got larger as the STB ratio increased, likely because the larger the STBs formed, the sparser the distribution of STBs became so that some of the randomly selected fields contained very few STBs. (H) Detection of gelatinase activities of MMP2 and MMP9 by gelatin zymography.
    Counterstaining With Dapi, supplied by Dojindo Labs, used in various techniques. Bioz Stars score: 96/100, based on 495 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/dapi+counterstain+solution/Cellstain+DAPI+solution/pmc11647102-473-2-6
    Average 96 stars, based on 495 article reviews
    counterstaining with dapi - by Bioz Stars, 2026-11
    96/100 stars

    Images

    1) Product Images from "Wnt signaling activation confers a syncytiotrophoblast progenitor state on trophoblast stem cells of cynomolgus monkey † "

    Article Title: Wnt signaling activation confers a syncytiotrophoblast progenitor state on trophoblast stem cells of cynomolgus monkey †

    Journal: Biology of Reproduction

    doi: 10.1093/biolre/ioae131

    Activation of Wnt/β-catenin signaling enhances STB differentiation. (A) A scheme of the culture conditions. Undifferentiated macTSCs were cultured with [CHIR(+)] or without [CHIR(−)] CHIR99021 for 3 days. Some cells were induced to differentiate in the medium containing dbcAMP [CHIR(−) cAMP and CHIR(+) cAMP] for another 6 days. (B) The relative expression level of a vCTB marker, TEAD4 . (C) Phase contrast images of cells on day 9. Multinucleated STB is outlined by a red line. Scale bar = 100 μm. (D, G) The relative expression levels of STB (D) and EVT (G) markers. For C, D, and G, the mean values (±SD) of technical triplicates of biological duplicates, normalized by the expression of ACTB , are shown relative to those in CHIR(−) (arbitrarily set as 1). Different letters represent significant differences ( p < 0.05). (E) Detection of multinucleated STBs by immunofluorescence for E-cadherin, CG, and DAPI staining. Scale bar = 100 μm. (F) The mean intensity ± SD (%) of STB nuclei calculated from four randomly chosen fields of biological duplicates. Different letters represent significant differences ( p < 0.05). Note that error bars got larger as the STB ratio increased, likely because the larger the STBs formed, the sparser the distribution of STBs became so that some of the randomly selected fields contained very few STBs. (H) Detection of gelatinase activities of MMP2 and MMP9 by gelatin zymography.
    Figure Legend Snippet: Activation of Wnt/β-catenin signaling enhances STB differentiation. (A) A scheme of the culture conditions. Undifferentiated macTSCs were cultured with [CHIR(+)] or without [CHIR(−)] CHIR99021 for 3 days. Some cells were induced to differentiate in the medium containing dbcAMP [CHIR(−) cAMP and CHIR(+) cAMP] for another 6 days. (B) The relative expression level of a vCTB marker, TEAD4 . (C) Phase contrast images of cells on day 9. Multinucleated STB is outlined by a red line. Scale bar = 100 μm. (D, G) The relative expression levels of STB (D) and EVT (G) markers. For C, D, and G, the mean values (±SD) of technical triplicates of biological duplicates, normalized by the expression of ACTB , are shown relative to those in CHIR(−) (arbitrarily set as 1). Different letters represent significant differences ( p < 0.05). (E) Detection of multinucleated STBs by immunofluorescence for E-cadherin, CG, and DAPI staining. Scale bar = 100 μm. (F) The mean intensity ± SD (%) of STB nuclei calculated from four randomly chosen fields of biological duplicates. Different letters represent significant differences ( p < 0.05). Note that error bars got larger as the STB ratio increased, likely because the larger the STBs formed, the sparser the distribution of STBs became so that some of the randomly selected fields contained very few STBs. (H) Detection of gelatinase activities of MMP2 and MMP9 by gelatin zymography.

    Techniques Used: Activation Assay, Cell Culture, Expressing, Marker, Immunofluorescence, Staining, Zymography

    macTSCs acquire FSK responsiveness for STB differentiation by Wnt/β-catenin signaling activation. (A) A scheme of the macTSC culture conditions. Undifferentiated macTSCs were cultured with [CHIR(+)] or without [CHIR(−)] CHIR99021 for 3 days. Some cells were induced to differentiate in the medium containing forskolin (FSK) [CHIR(−) FSK and CHIR(+) FSK] for another 6 days. (B) Phase contrast images of cells on day 9. Multinucleated STB is indicated by a red line. Scale bar = 100 μm. (C) The relative expression levels of STB markers. The mean values (±SD) of technical triplicates of biological duplicates, normalized by the expression of ACTB , are shown relative to those of cells maintained under the CHIR(−) condition (arbitrarily set as 1). (D) Detection of multinucleated STB by immunofluorescence for E-cadherin, CG, and DAPI staining. Scale bar = 100 μm. (E) The mean intensity ± SD (%) of STB nuclei calculated from four randomly chosen fields of biological duplicates. Different letters represent significant differences ( p < 0.05). Note that error bars got larger as the STB ratio increased, likely because the larger the STBs formed, the sparser the distribution of STBs became so that some of the randomly selected fields contained very few STBs.
    Figure Legend Snippet: macTSCs acquire FSK responsiveness for STB differentiation by Wnt/β-catenin signaling activation. (A) A scheme of the macTSC culture conditions. Undifferentiated macTSCs were cultured with [CHIR(+)] or without [CHIR(−)] CHIR99021 for 3 days. Some cells were induced to differentiate in the medium containing forskolin (FSK) [CHIR(−) FSK and CHIR(+) FSK] for another 6 days. (B) Phase contrast images of cells on day 9. Multinucleated STB is indicated by a red line. Scale bar = 100 μm. (C) The relative expression levels of STB markers. The mean values (±SD) of technical triplicates of biological duplicates, normalized by the expression of ACTB , are shown relative to those of cells maintained under the CHIR(−) condition (arbitrarily set as 1). (D) Detection of multinucleated STB by immunofluorescence for E-cadherin, CG, and DAPI staining. Scale bar = 100 μm. (E) The mean intensity ± SD (%) of STB nuclei calculated from four randomly chosen fields of biological duplicates. Different letters represent significant differences ( p < 0.05). Note that error bars got larger as the STB ratio increased, likely because the larger the STBs formed, the sparser the distribution of STBs became so that some of the randomly selected fields contained very few STBs.

    Techniques Used: Activation Assay, Cell Culture, Expressing, Immunofluorescence, Staining

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    Article Title: 4-Phenylbutyric acid activates an NF-κB-Egr-1 axis to control myoblast proliferation and ECM gene expression.
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    Sampling:

    Article Title: Impact of Host α2-Antiplasmin Deficiency on Engraftment and the Morphological Maturation of Transplanted Neural Precursor Cells in the Mouse Brain.
    Article Snippet: .. To evaluate graft size, one series (sampling interval: 180 μm) from each animal was counterstained with 4′,6-diamidino-2-phenylindole (DAPI; 1 : 800 in PBS; D523; Dojindo), mounted on glass slides, and imaged using an all-in-one fluorescence microscope (BZ-8000; Keyence, Osaka, Japan). .. The GFP-positive area was quantified as graft area using ImageJ software program (National Institutes of Health, Bethesda, MD, U.S.A.), and graft volume was estimated by multiplying the sum of GFP-positive areas by the section interval (180 μm).

    Glass:

    Article Title: Impact of Host α2-Antiplasmin Deficiency on Engraftment and the Morphological Maturation of Transplanted Neural Precursor Cells in the Mouse Brain.
    Article Snippet: .. To evaluate graft size, one series (sampling interval: 180 μm) from each animal was counterstained with 4′,6-diamidino-2-phenylindole (DAPI; 1 : 800 in PBS; D523; Dojindo), mounted on glass slides, and imaged using an all-in-one fluorescence microscope (BZ-8000; Keyence, Osaka, Japan). .. The GFP-positive area was quantified as graft area using ImageJ software program (National Institutes of Health, Bethesda, MD, U.S.A.), and graft volume was estimated by multiplying the sum of GFP-positive areas by the section interval (180 μm).

    Article Title: Dysferlin promotes inflammatory macrophage responses and modulates Ca 2+ -dependent STAT1 signaling.
    Article Snippet: .. After washing with PBS, the cells and sections were incubated with an Alexa Fluor 488 Goat Anti-Rabbit IgG H&L (ab150077; Abcam), Alexa Fluor 488 Goat Anti-Mouse IgG H&L (ab150113; Abcam), Alexa Fluor 594 Goat Anti-Rabbit IgG H&L (ab150080; Abcam), or an Alexa 594 Anti-Mouse IgG antibody (ab150116; Abcam) as a secondary antibody at room temperature for 1 h. the cells and sections were enclosed using ProLong Glass Antifade Mountant (P36965; Thermo Fisher Scientific) after staining the nuclei with 4′,6-diamidino-2phenylindole (DAPI; D523; Dojindo) at room temperature for 5 min. .. The fluorescent images were obtained under a fluorescence microscope (Keyence).

    Fluorescence:

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    Microscopy:

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    Article Snippet: .. To evaluate graft size, one series (sampling interval: 180 μm) from each animal was counterstained with 4′,6-diamidino-2-phenylindole (DAPI; 1 : 800 in PBS; D523; Dojindo), mounted on glass slides, and imaged using an all-in-one fluorescence microscope (BZ-8000; Keyence, Osaka, Japan). .. The GFP-positive area was quantified as graft area using ImageJ software program (National Institutes of Health, Bethesda, MD, U.S.A.), and graft volume was estimated by multiplying the sum of GFP-positive areas by the section interval (180 μm).

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    Staining:

    Article Title: Novel compounds induce AChR clustering and improve motor function in myasthenia gravis animal models.
    Article Snippet: The neuromuscular junction (NMJ), the synapse between motor nerves and skeletal muscles, is implicated in the pathogenesis of various neuromuscular disorders, including myasthenia gravis (MG), congenital myasthenia, and amyotrophic lateral sclerosis.. Postsynaptic clustering of acetylcholine receptors (AChRs) is crucial for NMJ formation.. This process is initiated by agrin, a glycoprotein secreted from motor nerve terminals, which activates muscle-specific tyrosine kinase (MuSK) via its coreceptor, low-density lipoprotein receptor-related protein 4 (Lrp4), and thereby activates downstream signaling pathways that induce AChR clustering.

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    Article Title: Dysferlin promotes inflammatory macrophage responses and modulates Ca 2+ -dependent STAT1 signaling.
    Article Snippet: .. After washing with PBS, the cells and sections were incubated with an Alexa Fluor 488 Goat Anti-Rabbit IgG H&L (ab150077; Abcam), Alexa Fluor 488 Goat Anti-Mouse IgG H&L (ab150113; Abcam), Alexa Fluor 594 Goat Anti-Rabbit IgG H&L (ab150080; Abcam), or an Alexa 594 Anti-Mouse IgG antibody (ab150116; Abcam) as a secondary antibody at room temperature for 1 h. the cells and sections were enclosed using ProLong Glass Antifade Mountant (P36965; Thermo Fisher Scientific) after staining the nuclei with 4′,6-diamidino-2phenylindole (DAPI; D523; Dojindo) at room temperature for 5 min. .. The fluorescent images were obtained under a fluorescence microscope (Keyence).

    Incubation:

    Article Title: Dysferlin promotes inflammatory macrophage responses and modulates Ca 2+ -dependent STAT1 signaling.
    Article Snippet: .. After washing with PBS, the cells and sections were incubated with an Alexa Fluor 488 Goat Anti-Rabbit IgG H&L (ab150077; Abcam), Alexa Fluor 488 Goat Anti-Mouse IgG H&L (ab150113; Abcam), Alexa Fluor 594 Goat Anti-Rabbit IgG H&L (ab150080; Abcam), or an Alexa 594 Anti-Mouse IgG antibody (ab150116; Abcam) as a secondary antibody at room temperature for 1 h. the cells and sections were enclosed using ProLong Glass Antifade Mountant (P36965; Thermo Fisher Scientific) after staining the nuclei with 4′,6-diamidino-2phenylindole (DAPI; D523; Dojindo) at room temperature for 5 min. .. The fluorescent images were obtained under a fluorescence microscope (Keyence).

    Article Title: Functional Characterization and Genotype‐Phenotype Correlation of Androgen Receptor Variants in Patients With Androgen Insensitivity Syndrome
    Article Snippet: .. After 48 h, cells were fixed with 4% paraformaldehyde for 15 min and blocked with 0.5% Triton X‐100‐containing blocking buffer for 2 h. Cells were incubated overnight with a primary anti‐AR antibody (Cell Signaling Technology, D6F11), followed by incubation with a fluorescent secondary antibody (Invitrogen, A21428) for 2 h. Nuclei were counterstained with DAPI (Dojindo Laboratories, D523) for 10 min. .. Images were acquired using a confocal laser‐scanning microscope (Zeiss) with a 40× objective.

    Blocking Assay:

    Article Title: Functional Characterization and Genotype‐Phenotype Correlation of Androgen Receptor Variants in Patients With Androgen Insensitivity Syndrome
    Article Snippet: .. After 48 h, cells were fixed with 4% paraformaldehyde for 15 min and blocked with 0.5% Triton X‐100‐containing blocking buffer for 2 h. Cells were incubated overnight with a primary anti‐AR antibody (Cell Signaling Technology, D6F11), followed by incubation with a fluorescent secondary antibody (Invitrogen, A21428) for 2 h. Nuclei were counterstained with DAPI (Dojindo Laboratories, D523) for 10 min. .. Images were acquired using a confocal laser‐scanning microscope (Zeiss) with a 40× objective.

    Cell-Signaling:

    Article Title: Functional Characterization and Genotype‐Phenotype Correlation of Androgen Receptor Variants in Patients With Androgen Insensitivity Syndrome
    Article Snippet: .. After 48 h, cells were fixed with 4% paraformaldehyde for 15 min and blocked with 0.5% Triton X‐100‐containing blocking buffer for 2 h. Cells were incubated overnight with a primary anti‐AR antibody (Cell Signaling Technology, D6F11), followed by incubation with a fluorescent secondary antibody (Invitrogen, A21428) for 2 h. Nuclei were counterstained with DAPI (Dojindo Laboratories, D523) for 10 min. .. Images were acquired using a confocal laser‐scanning microscope (Zeiss) with a 40× objective.



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    Activation of Wnt/β-catenin signaling enhances STB differentiation. (A) A scheme of the culture conditions. Undifferentiated macTSCs were cultured with [CHIR(+)] or without [CHIR(−)] CHIR99021 for 3 days. Some cells were induced to differentiate in the medium containing dbcAMP [CHIR(−) cAMP and CHIR(+) cAMP] for another 6 days. (B) The relative expression level of a vCTB marker, TEAD4 . (C) Phase contrast images of cells on day 9. Multinucleated STB is outlined by a red line. Scale bar = 100 μm. (D, G) The relative expression levels of STB (D) and EVT (G) markers. For C, D, and G, the mean values (±SD) of technical triplicates of biological duplicates, normalized by the expression of ACTB , are shown relative to those in CHIR(−) (arbitrarily set as 1). Different letters represent significant differences ( p < 0.05). (E) Detection of multinucleated STBs by immunofluorescence for E-cadherin, CG, and <t>DAPI</t> staining. Scale bar = 100 μm. (F) The mean intensity ± SD (%) of STB nuclei calculated from four randomly chosen fields of biological duplicates. Different letters represent significant differences ( p < 0.05). Note that error bars got larger as the STB ratio increased, likely because the larger the STBs formed, the sparser the distribution of STBs became so that some of the randomly selected fields contained very few STBs. (H) Detection of gelatinase activities of MMP2 and MMP9 by gelatin zymography.
    Dapi Based Mounting Solution Dapi I Counterstain 1000 Ng/Ml, supplied by Abbott Laboratories, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Activation of Wnt/β-catenin signaling enhances STB differentiation. (A) A scheme of the culture conditions. Undifferentiated macTSCs were cultured with [CHIR(+)] or without [CHIR(−)] CHIR99021 for 3 days. Some cells were induced to differentiate in the medium containing dbcAMP [CHIR(−) cAMP and CHIR(+) cAMP] for another 6 days. (B) The relative expression level of a vCTB marker, TEAD4 . (C) Phase contrast images of cells on day 9. Multinucleated STB is outlined by a red line. Scale bar = 100 μm. (D, G) The relative expression levels of STB (D) and EVT (G) markers. For C, D, and G, the mean values (±SD) of technical triplicates of biological duplicates, normalized by the expression of ACTB , are shown relative to those in CHIR(−) (arbitrarily set as 1). Different letters represent significant differences ( p < 0.05). (E) Detection of multinucleated STBs by immunofluorescence for E-cadherin, CG, and <t>DAPI</t> staining. Scale bar = 100 μm. (F) The mean intensity ± SD (%) of STB nuclei calculated from four randomly chosen fields of biological duplicates. Different letters represent significant differences ( p < 0.05). Note that error bars got larger as the STB ratio increased, likely because the larger the STBs formed, the sparser the distribution of STBs became so that some of the randomly selected fields contained very few STBs. (H) Detection of gelatinase activities of MMP2 and MMP9 by gelatin zymography.
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    Activation of Wnt/β-catenin signaling enhances STB differentiation. (A) A scheme of the culture conditions. Undifferentiated macTSCs were cultured with [CHIR(+)] or without [CHIR(−)] CHIR99021 for 3 days. Some cells were induced to differentiate in the medium containing dbcAMP [CHIR(−) cAMP and CHIR(+) cAMP] for another 6 days. (B) The relative expression level of a vCTB marker, TEAD4 . (C) Phase contrast images of cells on day 9. Multinucleated STB is outlined by a red line. Scale bar = 100 μm. (D, G) The relative expression levels of STB (D) and EVT (G) markers. For C, D, and G, the mean values (±SD) of technical triplicates of biological duplicates, normalized by the expression of ACTB , are shown relative to those in CHIR(−) (arbitrarily set as 1). Different letters represent significant differences ( p < 0.05). (E) Detection of multinucleated STBs by immunofluorescence for E-cadherin, CG, and <t>DAPI</t> staining. Scale bar = 100 μm. (F) The mean intensity ± SD (%) of STB nuclei calculated from four randomly chosen fields of biological duplicates. Different letters represent significant differences ( p < 0.05). Note that error bars got larger as the STB ratio increased, likely because the larger the STBs formed, the sparser the distribution of STBs became so that some of the randomly selected fields contained very few STBs. (H) Detection of gelatinase activities of MMP2 and MMP9 by gelatin zymography.
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    Image Search Results


    Age-dependent impairment in myelin phagocytosis and lipid droplet clearance in vitro and in vivo (Ai-ii) In vitro myelin phagocytosis assay with primary peritoneal macrophages from young (1-month-old) and aged (12-month-old) mice. (Ai) Representative immunofluorescence cytochemistry images (myelin basic protein (MBP, green), Iba1 (red), and DAPI (blue) of macrophages from young and aged mice incubated with myelin debris for 2, 4, 16, or 24 h. Scale bar: 20μm. (Aii) Quantification of macrophages containing phagocytosed myelin (% of MBP-positive Iba1- macrophages) at early (2–4 h) and late (16–24 h) time points. (Bi-iii) Delayed metabolism of phagocytosed myelin by aged macrophages. (Bi) Experimental scheme showing removal of myelin from culture medium after 16 h, and assessment of intracellular myelin metabolism by accumulation of Oil-Red-O (ORO) lipid droplets at 24 and 48 h. As an experimental control, representative images of MBP (green), Iba1 (red), and DAPI (blue) (Scale bar: 20μm) in young and aged macrophages showing comparable levels of phagocytosed MBP-immunoreactive myelin in young and aged macrophages at 16 h, and absence of MBP-immunoreactive myelin at 24 h and 48 h, (Bii) ORO and hematoxylin staining of young and aged macrophages showing ORO+ lipid droplet accumulation (red, arrowheads). (Biii) Quantification of ORO+ lipid–containing macrophages following myelin incubation and withdrawal, demonstrating impaired lipid clearance in aged compared to young macrophages. Scale bar: 3μm. (Ci-iii) In vivo accumulation of ORO+ lipid droplets in young (2-month-old) and old (24-month-old) mice during cuprizone-induced demyelination. (Ci) Representative ORO-stained coronal brain sections from young and aged mice after 4 (CPZ4), 6 (CPZ6) and 8 (CPZ8) weeks of continuous cuprizone feeding, and CPZ6 followed by 4 weeks of recovery after cuprizone withdrawal (CPZ6+4). Scale bar: 10μm. Quantification of ORO+ lipid droplets in, (Cii) the corpus callosum (CC) and (Ciii) dorsal fornix (DF) between young and aged mice at CPZ0, CPZ4, CPZ6, CPZ6+4 and CPZ8. (n = 5 mice/group; Mean ± SEM, p<0.05, *p<0.01, **p<0.001, ***p<0.0001).

    Journal: bioRxiv

    Article Title: Age-related loss of brain demyelinating and remyelinating potential is overcome by microglia renewal

    doi: 10.64898/2026.06.15.732390

    Figure Lengend Snippet: Age-dependent impairment in myelin phagocytosis and lipid droplet clearance in vitro and in vivo (Ai-ii) In vitro myelin phagocytosis assay with primary peritoneal macrophages from young (1-month-old) and aged (12-month-old) mice. (Ai) Representative immunofluorescence cytochemistry images (myelin basic protein (MBP, green), Iba1 (red), and DAPI (blue) of macrophages from young and aged mice incubated with myelin debris for 2, 4, 16, or 24 h. Scale bar: 20μm. (Aii) Quantification of macrophages containing phagocytosed myelin (% of MBP-positive Iba1- macrophages) at early (2–4 h) and late (16–24 h) time points. (Bi-iii) Delayed metabolism of phagocytosed myelin by aged macrophages. (Bi) Experimental scheme showing removal of myelin from culture medium after 16 h, and assessment of intracellular myelin metabolism by accumulation of Oil-Red-O (ORO) lipid droplets at 24 and 48 h. As an experimental control, representative images of MBP (green), Iba1 (red), and DAPI (blue) (Scale bar: 20μm) in young and aged macrophages showing comparable levels of phagocytosed MBP-immunoreactive myelin in young and aged macrophages at 16 h, and absence of MBP-immunoreactive myelin at 24 h and 48 h, (Bii) ORO and hematoxylin staining of young and aged macrophages showing ORO+ lipid droplet accumulation (red, arrowheads). (Biii) Quantification of ORO+ lipid–containing macrophages following myelin incubation and withdrawal, demonstrating impaired lipid clearance in aged compared to young macrophages. Scale bar: 3μm. (Ci-iii) In vivo accumulation of ORO+ lipid droplets in young (2-month-old) and old (24-month-old) mice during cuprizone-induced demyelination. (Ci) Representative ORO-stained coronal brain sections from young and aged mice after 4 (CPZ4), 6 (CPZ6) and 8 (CPZ8) weeks of continuous cuprizone feeding, and CPZ6 followed by 4 weeks of recovery after cuprizone withdrawal (CPZ6+4). Scale bar: 10μm. Quantification of ORO+ lipid droplets in, (Cii) the corpus callosum (CC) and (Ciii) dorsal fornix (DF) between young and aged mice at CPZ0, CPZ4, CPZ6, CPZ6+4 and CPZ8. (n = 5 mice/group; Mean ± SEM, p<0.05, *p<0.01, **p<0.001, ***p<0.0001).

    Article Snippet: This was followed by a three-probe amplification steps (AMP1-AMP3), fluorophore conjugation (Opal 570) and DAPI counterstaining (ACD, CA).

    Techniques: In Vitro, In Vivo, Phagocytosis Assay, Immunofluorescence, Incubation, Control, Staining

    Activation of Wnt/β-catenin signaling enhances STB differentiation. (A) A scheme of the culture conditions. Undifferentiated macTSCs were cultured with [CHIR(+)] or without [CHIR(−)] CHIR99021 for 3 days. Some cells were induced to differentiate in the medium containing dbcAMP [CHIR(−) cAMP and CHIR(+) cAMP] for another 6 days. (B) The relative expression level of a vCTB marker, TEAD4 . (C) Phase contrast images of cells on day 9. Multinucleated STB is outlined by a red line. Scale bar = 100 μm. (D, G) The relative expression levels of STB (D) and EVT (G) markers. For C, D, and G, the mean values (±SD) of technical triplicates of biological duplicates, normalized by the expression of ACTB , are shown relative to those in CHIR(−) (arbitrarily set as 1). Different letters represent significant differences ( p < 0.05). (E) Detection of multinucleated STBs by immunofluorescence for E-cadherin, CG, and DAPI staining. Scale bar = 100 μm. (F) The mean intensity ± SD (%) of STB nuclei calculated from four randomly chosen fields of biological duplicates. Different letters represent significant differences ( p < 0.05). Note that error bars got larger as the STB ratio increased, likely because the larger the STBs formed, the sparser the distribution of STBs became so that some of the randomly selected fields contained very few STBs. (H) Detection of gelatinase activities of MMP2 and MMP9 by gelatin zymography.

    Journal: Biology of Reproduction

    Article Title: Wnt signaling activation confers a syncytiotrophoblast progenitor state on trophoblast stem cells of cynomolgus monkey †

    doi: 10.1093/biolre/ioae131

    Figure Lengend Snippet: Activation of Wnt/β-catenin signaling enhances STB differentiation. (A) A scheme of the culture conditions. Undifferentiated macTSCs were cultured with [CHIR(+)] or without [CHIR(−)] CHIR99021 for 3 days. Some cells were induced to differentiate in the medium containing dbcAMP [CHIR(−) cAMP and CHIR(+) cAMP] for another 6 days. (B) The relative expression level of a vCTB marker, TEAD4 . (C) Phase contrast images of cells on day 9. Multinucleated STB is outlined by a red line. Scale bar = 100 μm. (D, G) The relative expression levels of STB (D) and EVT (G) markers. For C, D, and G, the mean values (±SD) of technical triplicates of biological duplicates, normalized by the expression of ACTB , are shown relative to those in CHIR(−) (arbitrarily set as 1). Different letters represent significant differences ( p < 0.05). (E) Detection of multinucleated STBs by immunofluorescence for E-cadherin, CG, and DAPI staining. Scale bar = 100 μm. (F) The mean intensity ± SD (%) of STB nuclei calculated from four randomly chosen fields of biological duplicates. Different letters represent significant differences ( p < 0.05). Note that error bars got larger as the STB ratio increased, likely because the larger the STBs formed, the sparser the distribution of STBs became so that some of the randomly selected fields contained very few STBs. (H) Detection of gelatinase activities of MMP2 and MMP9 by gelatin zymography.

    Article Snippet: After nuclear counterstaining with DAPI (D523; DOJINDO, Kumamoto, Japan), the cells were mounted on glass slides with VECTASHIELD (H-1000; Vector Laboratories, Newark, CA, USA).

    Techniques: Activation Assay, Cell Culture, Expressing, Marker, Immunofluorescence, Staining, Zymography

    macTSCs acquire FSK responsiveness for STB differentiation by Wnt/β-catenin signaling activation. (A) A scheme of the macTSC culture conditions. Undifferentiated macTSCs were cultured with [CHIR(+)] or without [CHIR(−)] CHIR99021 for 3 days. Some cells were induced to differentiate in the medium containing forskolin (FSK) [CHIR(−) FSK and CHIR(+) FSK] for another 6 days. (B) Phase contrast images of cells on day 9. Multinucleated STB is indicated by a red line. Scale bar = 100 μm. (C) The relative expression levels of STB markers. The mean values (±SD) of technical triplicates of biological duplicates, normalized by the expression of ACTB , are shown relative to those of cells maintained under the CHIR(−) condition (arbitrarily set as 1). (D) Detection of multinucleated STB by immunofluorescence for E-cadherin, CG, and DAPI staining. Scale bar = 100 μm. (E) The mean intensity ± SD (%) of STB nuclei calculated from four randomly chosen fields of biological duplicates. Different letters represent significant differences ( p < 0.05). Note that error bars got larger as the STB ratio increased, likely because the larger the STBs formed, the sparser the distribution of STBs became so that some of the randomly selected fields contained very few STBs.

    Journal: Biology of Reproduction

    Article Title: Wnt signaling activation confers a syncytiotrophoblast progenitor state on trophoblast stem cells of cynomolgus monkey †

    doi: 10.1093/biolre/ioae131

    Figure Lengend Snippet: macTSCs acquire FSK responsiveness for STB differentiation by Wnt/β-catenin signaling activation. (A) A scheme of the macTSC culture conditions. Undifferentiated macTSCs were cultured with [CHIR(+)] or without [CHIR(−)] CHIR99021 for 3 days. Some cells were induced to differentiate in the medium containing forskolin (FSK) [CHIR(−) FSK and CHIR(+) FSK] for another 6 days. (B) Phase contrast images of cells on day 9. Multinucleated STB is indicated by a red line. Scale bar = 100 μm. (C) The relative expression levels of STB markers. The mean values (±SD) of technical triplicates of biological duplicates, normalized by the expression of ACTB , are shown relative to those of cells maintained under the CHIR(−) condition (arbitrarily set as 1). (D) Detection of multinucleated STB by immunofluorescence for E-cadherin, CG, and DAPI staining. Scale bar = 100 μm. (E) The mean intensity ± SD (%) of STB nuclei calculated from four randomly chosen fields of biological duplicates. Different letters represent significant differences ( p < 0.05). Note that error bars got larger as the STB ratio increased, likely because the larger the STBs formed, the sparser the distribution of STBs became so that some of the randomly selected fields contained very few STBs.

    Article Snippet: After nuclear counterstaining with DAPI (D523; DOJINDO, Kumamoto, Japan), the cells were mounted on glass slides with VECTASHIELD (H-1000; Vector Laboratories, Newark, CA, USA).

    Techniques: Activation Assay, Cell Culture, Expressing, Immunofluorescence, Staining