oil red o staining modified oil red o staining kit  (Beyotime)

Journal: Bioactive Materials

Article Title: Sulfated polysaccharide prevents senescent adipocyte-driven osteonecrosis by stem cell fate reprogramming

doi: 10.1016/j.bioactmat.2025.11.039

Figure Lengend Snippet: SCS suppresses senescence cascade amplification by attenuating secondary spread from GC-induced primary senescent adipocytes. ( A ) Schematic illustration of SCS intervention exclusively during the fully developed senescent phase of MPS-induced bone marrow. ( B ) qPCR analysis of senescence-associated markers ( Cdkn1b , Cdkn1a , and Cdkn2c ) in bone tissues at 4 weeks following combined SCS and MPS treatment. n = 3 biological replicates. ( C ) ELISA analysis of bone marrow senescence-associated factors (IL-1β, IL-18, TNF-α, IL-6, CXCL1, and CCL3) after 4 weeks of combined treatment with SCS and MPS. n = 4 biological replicates. ( D ) Quantification of the maximal compressive load of the isolated distal femur and femoral diaphysis. n = 6 biological replicates. ( E ) Schematic diagram depicting isolation of bone marrow adipocytes from mice treated with SCS and MPS for 14 days using mature adipocyte-specific fast centrifugation and construction of a senescence propagation model in vitro . ( F and G ) Representative flow cytometry plots (D) and quantification (E) of EdU-positive (proliferating) CD45 − Ter119 − CD31 − LepR + MSCs cultured for 3 days with adipocyte conditioned medium (CM). n = 6 biological replicates. ( H and I ) Representative ALP staining images (F) and corresponding quantification of ALP activity (G) in CD45 − Ter119 − CD31 − LepR + MSCs cultured with SCS-induced adipocyte CM. n = 6 biological replicates. (Scale bars, 50 μm and 30 μm) ( J and K ) Representative Oil Red O staining (H) and quantification (I) of adipogenic differentiation in MSCs cultured with SCS-induced adipocyte CM. n = 6 biological replicates. (Scale bars, 50 μm and 25 μm) ( L and M ) Representative images (J) and quantification (K) of crystal violet-stained fibroblast colony-forming units (CFU-F) in MSCs cultured with various adipocyte CMs. n = 6 biological replicates. (Scale bars, 400 μm) ( N ) qPCR analysis of senescence-related markers ( Cdkn2a and Cdkn1a ) in MSCs treated with different adipocyte CMs. n = 3 biological replicates. ( O and P ) Representative immunofluorescence-FISH images (M) and quantification (N) showing colocalization of γ-H2A.X with telomere-associated foci (TAF) in MSCs cultured with different adipocyte CMs. n = 6 biological replicates. (Scale bars, 7 μm and 1 μm) ( Q and R ) Representative images (O) and quantification (P) of 2D tube formation assays in HUVECs cultured for 3 days with various adipocyte CMs. n = 6 biological replicates. (Scale bars, 100 μm and 25 μm) ( S and T ) Representative images (Q) and quantification (R) of SA-β-Gal–positive HUVECs (green) following 3-day treatment with different adipocyte CMs. n = 6 biological replicates. (Scale bars, 100 μm and 25 μm) ( U ) qPCR analysis of the senescence-related gene LMNB1 in HUVECs treated with various adipocyte CMs. n = 3 biological replicates. Data are presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant. Statistical significance was determined using an unpaired two-tailed Student's t -test ( B, C, D, G, I, K, M, N, R, T and U ).

Article Snippet: Afterward, the cells were fixed with 70 % ethanol for 15 min and stained for intracellular lipid droplets using an Oil Red O Staining Kit (Servicebio, G1015) according to the manufacturer's instructions.

Techniques: Amplification, Enzyme-linked Immunosorbent Assay, Isolation, Centrifugation, In Vitro, Flow Cytometry, Cell Culture, Staining, Activity Assay, Immunofluorescence, Two Tailed Test

Journal: Bioactive Materials

Article Title: Sulfated polysaccharide prevents senescent adipocyte-driven osteonecrosis by stem cell fate reprogramming

doi: 10.1016/j.bioactmat.2025.11.039

Figure Lengend Snippet: SCS modulates mesenchymal stem cell lineage bias via activation of the IGF-1/PI3K/Akt/mTOR signaling pathway. ( A ) Quantitative analysis of osteocyte morphology in the trabecular bone matrix of the bone marrow at week 6 after MPS treatment with or without SCS, in the presence of various neutralizing antibodies (NAbs) and antagonistic proteins. ( B ) ELISA analysis of IGF-1 and BMP-2 levels in the femoral bone marrow and peripheral serum at day 7 following SCS treatment under MPS conditions. ( C and D ) Western blot analysis of phospho-PI3K, phospho-Akt, and phospho-mTOR (C), as well as phospho-Smad1/5/8, phospho-ERK, and phospho-p38 (D), in CD45 − Ter119 − CD31 − LepR + MSCs after 15-min stimulation with conditioned medium (CM) derived from bone marrow fluid at day 7 following SCS treatment. ( E – G ) Representative flow cytometry plots (E, F) and quantitative analysis (G) of CD45 − CD31 − Sca-1 + CD24 − adipocyte progenitor cells (APCs), CD45 − CD31 − Sca-1 + CD24 + MSCs (E), and CD45 − CD31 − Sca-1 − PDGFRα + (Pα + ) osteoprogenitor cells (OPCs) (F) from femoral bone marrow at day 14 post-MPS induction with or without combined treatment using SCS and IGF-1 NAb or Noggin. ( H and I ) Representative SA-β-Gal staining images (green) of the femur (H), and corresponding quantification (I), at week 4 following MPS treatment with SCS in combination with IGF-1 NAb or DMH1. Insets show magnified views of bone marrow (BM) and trabecular bone matrix (TBM) regions. (Scale bars, 100 μm and 25 μm) ( J ) qPCR analysis of 12 senescence-associated markers in ex vivo femoral bone tissues at week 4 following MPS treatment with SCS in combination with IGF-1 NAb or DMH1. ( K ) Representative Oil Red O staining images of CD45 − Ter119 − CD31 − LepR + MSCs sorted from femurs at day 7 following MPS treatment with SCS in combination with LY294002 or LDN-193189, after in vitro adipogenic induction. (Scale bars, 50 μm and 25 μm) ( L and M ) γ-H2A.X and telomere-associated DNA damage foci (TAFs) co-localization analysis (L), and corresponding quantification (M), in CD45 − Ter119 − CD31 + arteriolar ECs sorted from femurs at day 28 following MPS treatment with SCS in combination with rapamycin or LDN-193189, using immuno-FISH staining. (Scale bars, 7 μm and 1 μm) ( N and O ) Sequential fluorescent labeling using calcein (N) and quantification of mineral apposition rate (O) in femurs treated with SCS and MPS for 4 weeks, with or without LY294002 and/or GW9662. (Scale bars, 50 μm) ( P ) ELISA analysis of five senescence-associated cytokines in femoral bone marrow at day 28 following MPS treatment with SCS in combination with rapamycin and/or T0070907. ( Q and R ) Representative t-distributed stochastic neighbor embedding (t-SNE) plots (Q) from flow cytometric analysis of CD45 − CD31 − Sca-1 + CD24 − APCs, CD45 − CD31 − Sca-1 + CD24 + MSCs, CD45 − CD31 − Sca-1 − Pα + OPCs, CD45 − Ter119 − CD31 + arteriolar ECs, and CD45 − Ter119 − Emcn + sinusoidal ECs at day 14 following MPS treatment with SCS in combination with IGF-1 and/or rosiglitazone, and quantitative analysis of APCs (R) ( S ) Heatmap showing the fluorescent intensity distribution of Lamin-B1 expression across five cellular subpopulations as identified in the t-SNE clustering plot. ∗ P < 0.05 vs. IgG (empty lacunae); # P < 0.05 vs. IgG (filled lacunae). ∗ P < 0.05 vs. SCS; # P < 0.05 vs. SCS + IGF-1 NAb. Data are presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant. Statistical significance was determined using an unpaired two-tailed Student's t -test ( B ), or one-way ANOVA with Tukey's post hoc test ( A, G, I, J, O, P and R ).

Article Snippet: Afterward, the cells were fixed with 70 % ethanol for 15 min and stained for intracellular lipid droplets using an Oil Red O Staining Kit (Servicebio, G1015) according to the manufacturer's instructions.

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Derivative Assay, Flow Cytometry, Staining, Ex Vivo, In Vitro, Labeling, Expressing, Two Tailed Test

Journal: Redox Biology

Article Title: Hyodeoxycholic acid attenuates atherosclerosis by antagonizing FXR and modulating the PD-1/mTORC1 signaling axis

doi: 10.1016/j.redox.2026.104096

Figure Lengend Snippet: AS was associated with decreased serum levels of HDCA and impaired Treg accumulation within vascular plaques. (A) Serum HDCA levels were reduced in AS patients relative to healthy individuals. The observed overlap reflects expected biological variability in human populations. (B) A negative correlation was observed between serum HDCA concentration and disease activity scores in AS patients. (C) Histopathological analysis of arterial samples revealed that higher HDCA levels correlated with reduced arterial intima thickness and plaque formation (magnification, 2 × ; scale bar, 1 mm). (D) Confocal microscopy showed increased infiltration of Foxp3 + Tregs (red) in AS patients with higher HDCA levels, whereas the percentages of CD11c + cells (red) did not significantly differ between groups. CD25 + cells (green, in panel A) and MHC-II + cells (green, in panel B) are presented for additional immunofluorescent phenotyping. Cell nuclei were stained with DAPI (blue). Representative images are shown (magnification, 10 × ; scale bar, 100 μm). (E) Serum HDCA concentrations are significantly decreased in AS mouse models. (F) Oil Red O staining of arterial sections showing reduced lipid deposition following HDCA treatment. (G) Assessment of hemodynamic parameters, including systolic and diastolic blood pressure (SBP and DBP) and heart rate. (H) Oil Red O and immunohistochemical analysis of lesions area and IFN-γ expression in arterial section (magnification, 2 × ; scale bar, 1.25 mm). (I) Flow cytometry of PKH26-labeled donor Tregs revealed that HDCA treatment selectively increased Treg accumulation in atherosclerotic plaques, with no effect in spleen tissue. (J) Quantification of Foxp3 + Tregs (red), CD45 + leukocytes (red), CD68 + macrophages (red), and GR1 + monocytes/granulocytes (red) in atherosclerotic plaques following HDCA treatment; approximately 500 cells were counted per group. CD25 + cells (green), CD3 + T cells (green), F4/80 + macrophages (green), and Ly6G + neutrophils (green) were visualized by immunofluorescence co-staining. Cell nuclei were labeled with DAPI (blue). (K – L) ELISA quantification of serum IL-6 (K) and TNF-α (L) levels in AS mice with or without HDCA treatment. (M) IL-35 levels in the supernatant of Treg cells were measured by ELISA after 48 h of culture with or without HDCA treatment. (N) Representative H&E staining of arterial sections from AS mice to evaluate atherosclerotic lesion area following HDCA or anti-CD25 treatment. Data are presented as mean ± SD (n = 5 biological replicates). Statistical significance was determined using two-tailed Student's t -test or one-way ANOVA. ns: not significant; ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.

Article Snippet: We used the Oil Red O staining kit (C0157S, Beyotime).

Techniques: Concentration Assay, Activity Assay, Confocal Microscopy, Staining, Immunohistochemical staining, Expressing, Flow Cytometry, Labeling, Immunofluorescence, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Redox Biology

Article Title: Hyodeoxycholic acid attenuates atherosclerosis by antagonizing FXR and modulating the PD-1/mTORC1 signaling axis

doi: 10.1016/j.redox.2026.104096

Figure Lengend Snippet: HDCA modulates Treg migration and atherosclerotic plaque composition via FXR signaling. ApoE−/− mice (C57BL/6J background, male, 8 weeks old) were fed a high-fat diet for 28 days to induce AS and subsequently received adoptive transfer of control or FXR-knockout (FXR KO) Treg cells generated by CRISPR/Cas9-mediated lentiviral transduction, with HDCA (30 μM) or vehicle treatment as indicated. (A) Representative Western blot analysis of FXR, PD-1, SHP-2, p-Raptor, RAC and IL-10R expression in isolated Treg cells from each group. (B) Oil Red O staining was performed to assess lipid accumulation in the aorta. (C) Masson's trichrome staining of aortic sections from FXR KO mice reveals comparable plaque area and collagen deposition in both HDCA-treated and untreated groups (magnification, 5 × ; scale bar, 1 mm). (D) Representative H&E images of aortic sections show the difference between untreated and HDCA-treated mice in the FXR KO groups (magnification, 5 × ; scale bar, 500 μm). Relative bar graphs show quantification of lesion area and lesion/media area ratio. (E) Confocal immunofluorescence was used to evaluate Foxp3+ Treg infiltration within atherosclerotic plaques (scale bar, 25 μm). (F) Immunohistochemistry images show Treg accumulation in the plaque area across all groups (magnification, 40 × ; scale bar, 100 μm). (G) Flow cytometry analysis of Treg proportions in aortic plaques and spleen from control and FXR KO mice, with or without HDCA treatment. (H) Western blot analysis of matrix remodeling-related proteins, including calpain 1 and matrix metalloproteinase 2, and the anti-inflammatory factor IL-10. Data are presented as mean ± SD (n = 5 biological replicates). Data with four groups were analyzed by one-way ANOVA with Tukey's post hoc test. Comparisons between two groups were performed using the non-parametric Mann-Whitney U test. ns, not significant; ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.

Article Snippet: We used the Oil Red O staining kit (C0157S, Beyotime).

Techniques: Migration, Adoptive Transfer Assay, Control, Knock-Out, Generated, CRISPR, Transduction, Western Blot, Expressing, Isolation, Staining, Immunofluorescence, Immunohistochemistry, Flow Cytometry, MANN-WHITNEY

Journal: Redox Biology

Article Title: Hyodeoxycholic acid attenuates atherosclerosis by antagonizing FXR and modulating the PD-1/mTORC1 signaling axis

doi: 10.1016/j.redox.2026.104096

Figure Lengend Snippet: PD-1 blockade promotes Treg migration via mTORC1-driven glycolytic pathways . (A) Representative en face images of Oil Red O-stained aortas from control and PD-1 blockade groups. (B) Representative images of arterial lesions stained with H&E (scale bar, 100 μm) and Oil Red O (scale bar, 200 μm), with quantification of the necrotic core area and Oil Red O-positive areas. (C) Flow cytometry detection of the enrichment of Foxp3+ Tregs in plaque areas following adoptive transfer of Tregs transduced with shRaptor or control vector, with or without PD-1 blockade. (D) Representative H&E staining of aortic sections showing the percentage of atherosclerotic plaque area relative to the total arterial area in each group (scale bar, 100 μm; magnification, 10 × ). (E) Kaplan-Meier survival analysis of skin grafts in recipients treated with control Tregs (pLKO.1), shRaptor Tregs, PD-1 blockade, or shRaptor plus PD-1 blockade. (F–I) Metabolic analysis of donor Tregs after shRaptor or PD-1 blockade, showing effects on glycolysis (ECAR) and mitochondrial respiration (OCR). (J – K) Western blot and flow cytometry analysis of CPT1a expression levels in Treg isolated from plaques to evaluate alterations in fatty acid oxidation pathways following PD-1 blockade. (L) Western blot analysis was used to assess pERK, ERK, pS6K, S6K, and RAC levels in Treg cells transduced with control shRNA (pLKO.1) or Raptor shRNA (shRaptor) following PD-1 blockade at 0, 15, and 30 min. Statistical analyses were performed using one-way ANOVA followed by Tukey's post-hoc test for multiple comparisons, and the log-rank test for survival analysis. Data are presented as mean ± SD (n = 3-5 biological replicates). Statistical significance is indicated as follows: ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.

Article Snippet: We used the Oil Red O staining kit (C0157S, Beyotime).

Techniques: Migration, Staining, Control, Flow Cytometry, Adoptive Transfer Assay, Transduction, Plasmid Preparation, Western Blot, Expressing, Isolation, shRNA

Journal: Redox Biology

Article Title: Hyodeoxycholic acid attenuates atherosclerosis by antagonizing FXR and modulating the PD-1/mTORC1 signaling axis

doi: 10.1016/j.redox.2026.104096

Figure Lengend Snippet: ZNF671 governs HDCA-driven STAT5 activation and metabolic adaptation in Treg cells. Ex vivo Tregs were transduced with shZNF671 or vector treated w/wo HDCA prior to iv-injection in recipient AS recipients. Tregs isolated from atherosclerotic plaque based on Foxp3 expression. (A) Flow cytometric analysis and quantification ZNF671 expression in Foxp3+ Tregs isolated from plaques in Vector and FXR KO groups ± HDCA. (B) Western blot analysis of protein expression levels of ZNF671, MAPK6, and SIAH1 in each group. (C) Quantification of atherosclerotic plaque area by percentage of Oil Red O staining-positive area in aorta from different groups. (D) Quantification of atherosclerotic lesion area in indicated groups. (E – F) ELISA was used to quantify the levels of anti-inflammatory cytokines IL-10 and IL-35 in culture supernatants of Treg cells from the indicated groups. (G) Migration of PKH26-labeled Treg cells was quantified by flow cytometry 24 h post-transfer. (H–I) Seahorse assay showed that OCR was significantly reduced in the ZNF671 KO group and this reduction was further exacerbated by HDCA treatment. (J) Immunofluorescence staining showed that ZNF671 KO enhanced pSTAT5 signaling, and this effect was significantly amplified by HDCA treatment. Quantification of pSTAT5 levels is shown in the bar graphs as mean ± SD from 500 cells per coverslip. scale bar, 25 μm. (K) Flow cytometry plots and quantification the percentage of pSTAT5+Foxp3+ Treg cells across all groups. Data are presented as the mean ± SD (n = 3-5 biological replicates). Statistical analysis was performed using one-way ANOVA followed by post-hoc tests. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.

Article Snippet: We used the Oil Red O staining kit (C0157S, Beyotime).

Techniques: Activation Assay, Ex Vivo, Transduction, Plasmid Preparation, IV Injection, Isolation, Expressing, Western Blot, Staining, Enzyme-linked Immunosorbent Assay, Migration, Labeling, Flow Cytometry, Immunofluorescence, Amplification