counterstaining with dapi  (Dojindo Labs)

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

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

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