media with dapi  (Vector Laboratories)

Journal: Cellular and Molecular Neurobiology

Article Title: Characterization of Perineuronal Nets in the Paraventricular Nucleus of the Hypothalamus and their Alteration in Neurogenic Hypertension

doi: 10.1007/s10571-025-01628-z

Figure Lengend Snippet: Developmental regulation of PNNs in the PVN. a WFA staining (magenta) shows no visible PNNs at P6, with progressive appearance at P14 and maturation by 1Mo. b-c Quantification of WFA+ area (Time, p < 0.0001; Sex, p = 0.1140; Interaction, p = 0.6546) and PNN intensity (Time, p < 0.0001; Sex, p = 0.0874; Interaction, p = 0.7332) across developmental timepoints (each data point = average of 3 mice, 7–12 sections per mouse). Two-way ANOVA with Tukey’s multiple comparisons. * p > 0.05 vs. P6 for each sex. PVN-F denotes females, and PVN-M denotes males. d Representative images of PNN-enwrapped neurons at P14, 3Mo, and 25Mo. e-f Quantification of WFA+ area (%) and fluorescence intensity in 3- vs. 25-month-old naïve male mice (3Mo n = 4; 25Mo n = 5). Data are shown as mean ± SEM; circles represent individual mice (average of 7–12 bilateral PVN sections/mouse). DAPI (blue) indicates nuclei and HuC/D (green) indicates neurons

Article Snippet: Sections were mounted using VECTASHIELD Hardset Antifade Mounting Media with DAPI (Vector Laboratories, H-1500-10).

Techniques: Staining, Fluorescence

Journal: Clinical and Experimental Immunology

Article Title: Senolytic treatment to rescue hallmarks of senescence in lymph node fibroblasts from patients with rheumatoid arthritis: Implications for premature aging and potential therapeutic intervention in early rheumatoid arthritis

doi: 10.1093/cei/uxaf029

Figure Lengend Snippet: morphological alterations in directly ex vivo analyzed and cultured RA LN fibroblasts compared with controls. (A) Flow cytometry gating strategy used to identify single cells (FSC-H vs. FSC-A), CD45 - CD235 - cells (SSC-A v.s CD45/CD235a), and CD45 - LN fibroblasts. Numbers adjacent to the outlined areas indicate the percentages of cells in the gated population. (B) Cell size and (C) cell granularity measured by flow cytometry. Green = non-kidney transplant healthy volunteers undergoing LN needle biopsy. (D) Representative phase-contrast images of cultured LN fibroblasts generated via IncuCyte ZOOM. Insert: representative immunofluorescent stainings of LN fibroblasts nuclei using DAPI. (E) Representative flow cytometry plots showing the LN fibroblast population on forward–sideward scatter. (F) Cell size measured by flow cytometry (MFI of FSC-W). (G) Cell granularity (MFI of SSC-A). (H) Nuclear size of cultured LN fibroblasts measured by immunofluorescent DAPI staining; approximately 50 cells per donor were measured for analysis via LAS X 3D and ImageJ. (I) Autofluorescence of unstained cells in the FITC channel. All donors were at passage 5 for flow cytometry and at passage 6 for nuclear size measurements. N = 5 per group. Data are presented as median + interquartile range. Statistical differences were determined using a Kruskal–Wallis test followed by Dunn’s multiple comparisons test.

Article Snippet: Coverslips were mounted on microscope slides with DAPI Vectashield Hardset mounting media (Vector Laboratories). yH2AX staining, which represents DNA damage at baseline, was analyzed using confocal microscopy (TCS SP8, Leica Microsystems).

Techniques: Ex Vivo, Cell Culture, Flow Cytometry, Generated, Staining

Journal: Clinical and Experimental Immunology

Article Title: Senolytic treatment to rescue hallmarks of senescence in lymph node fibroblasts from patients with rheumatoid arthritis: Implications for premature aging and potential therapeutic intervention in early rheumatoid arthritis

doi: 10.1093/cei/uxaf029

Figure Lengend Snippet: significantly higher SA-β-gal activity in RA-risk and RA LN fibroblasts compared with controls. (A) Representative image reflecting positive and negative SA-β-gal staining when the Senescence Counter macro in ImageJ was used. (B) Representative images of SA-β-gal (dark blue) combined with DAPI nuclear staining (light blue in cultured LN fibroblasts. C) SA-β-gal-positive LN fibroblasts as a percentage of DAPI-positive cells. All donors at passage 6, n = 5 per group and approximately 50 cells per donor, were analyzed. Data are presented as median + interquartile range. Statistical differences were determined using a Kruskal–Wallis test followed by Dunn’s multiple comparisons test.

Article Snippet: Coverslips were mounted on microscope slides with DAPI Vectashield Hardset mounting media (Vector Laboratories). yH2AX staining, which represents DNA damage at baseline, was analyzed using confocal microscopy (TCS SP8, Leica Microsystems).

Techniques: Activity Assay, Staining, Cell Culture

Journal: Clinical and Experimental Immunology

Article Title: Senolytic treatment to rescue hallmarks of senescence in lymph node fibroblasts from patients with rheumatoid arthritis: Implications for premature aging and potential therapeutic intervention in early rheumatoid arthritis

doi: 10.1093/cei/uxaf029

Figure Lengend Snippet: dasatinib treatment restores increased lysosomal accumulation in LN fibroblasts. (A) SA-β-gal-positive LN fibroblasts as a percentage of DAPI-positive cells before and after treatment with 5 µM dasatinib. (B) Relative gene expression level of GLB1. (C) Representative images of SenTraGor-stained lipofuscin granules (red) and DAPI-stained nuclei (blue) in cultured LN fibroblasts. (D) Percentage of SenTraGor-positive cells relative to DAPI-positive cells as measured by LAS X 3D and ImageJ. All donors at passage 6, N = 5 per group, approximately 50 cells per condition, were analyzed. Data are presented as median + interquartile range. Statistical differences at baseline were determined using a Kruskal–Wallis test, followed by Dunn’s multiple comparisons test and a Wilcoxon matched pairs signed-rank test was used to analyze the effect of dasatinib treatment.

Article Snippet: Coverslips were mounted on microscope slides with DAPI Vectashield Hardset mounting media (Vector Laboratories). yH2AX staining, which represents DNA damage at baseline, was analyzed using confocal microscopy (TCS SP8, Leica Microsystems).

Techniques: Gene Expression, Staining, Cell Culture

Journal: Clinical and Experimental Immunology

Article Title: Senolytic treatment to rescue hallmarks of senescence in lymph node fibroblasts from patients with rheumatoid arthritis: Implications for premature aging and potential therapeutic intervention in early rheumatoid arthritis

doi: 10.1093/cei/uxaf029

Figure Lengend Snippet: Impaired DNA damage repair in RA(-risk) LN fibroblasts. (A) Representative images of yH2AX foci (pink) and DAPI staining (blue) in cultured LN fibroblasts. (B) Average number of yH2AX foci per nucleus in cultured LN fibroblasts before and after 5 µM dasatinib treatment. All donors were at passage 6 under unstimulated conditions. (C) Representative images of yH2AX foci (pink) and DAPI staining (blue) in cultured LN fibroblasts after DNA damage induction by gamma-irradiation. (B) Average number of yH2AX foci per nucleus in cultured LN fibroblasts directly and 20 and 40 hours after irradiation. Mean value per donor was determined through the quantification of Z-stack images of approximately 50 cells per donor. All donors were at passage 7, N = 5 per group. Data are presented as median + interquartile range. Statistical differences were determined using two-way ANOVA + Dunnett’s T3 multiple comparisons test, and a Wilcoxon matched pairs signed-rank test was used to analyze the effect of dasatinib treatment.

Article Snippet: Coverslips were mounted on microscope slides with DAPI Vectashield Hardset mounting media (Vector Laboratories). yH2AX staining, which represents DNA damage at baseline, was analyzed using confocal microscopy (TCS SP8, Leica Microsystems).

Techniques: Staining, Cell Culture, Irradiation