nuclear counterstaining  (Beyotime)

Journal: International Journal of Biological Sciences

Article Title: Hypoxia Reprograms a Ciliary Scaffold into a Metabolic-Epigenetic Trafficking Hub via a Succinate-Driven DNMT3L-ASXL2 Axis

doi: 10.7150/ijbs.137355

Figure Lengend Snippet: Overexpressing CEP162 reduces sperm motility in mice. (A) Sperm viability and kinematic analysis following CEP162 overexpression under normoxia (21% O 2 ) and hypoxia (11.7% O 2 ). The sperm motility parameters, including progressive motility, VCL, VSL, VAP, STR, BCF, ALH, LIN, and WOB, were quantified using computer-assisted sperm analysis (CASA; Microptic S.L., Barcelona, Spain). (B) Ultrastructural analysis of sperm flagella by transmission electron microscopy. Representative micrographs show transverse sections of the flagellum, including the midpiece (with a mitochondrial sheath), principal piece (with a fibrous sheath), and end piece (showing axonemal termination). All scale bars, 200 nm. The lower panel shows the quantification of axonemal DMT defects (%). (C) Microtubule regrowth following nocodazole washout in CEP162-overexpressing G2 cells. The percentage of pericentrin-expressing cells was calculated by dividing the number of pericentrin-positive cells by the total number of cells in each respective group. (D) Targeted proteomic profiling of CEP162-overexpressing sperm flagella. Heatmap showing Z score-normalized expression of selected TEKT family members involved in sperm axoneme assembly and Rab GTPases across three biological replicates. t-SNE visualization of the correlations among TEKT1, TEKT2, TEKT3, TEKT4, TEKT5, EFHC1, Rab1a, RAB1b, and RAB14 expression in the sperm flagella of control and CEP162-overexpressing mice. (E) Western blot analysis of TEKT2 protein expression in whole sperm and flagella isolated from CEP162-overexpressing mice maintained under normoxia (21% O 2 ) or hypoxia (11.7% O 2 ) for 10 weeks. Note: This Western blot detected endogenous TEKT2 (~50 kDa); the RFP-TEKT2-FKBP fusion protein (~87 kDa) was used exclusively for immunofluorescence localization in Panels F-G. (F-G) Mice were injected with recombinant AAV9 vectors encoding RFP-TEKT2-FKBP (~87 kDa) and maintained under (F) 21% O₂ (normoxia) or (G) 11.7% O₂ (hypoxia) for 10 weeks. Sperm were collected from the cauda epididymis and subjected to triple-label immunofluorescence for RFP (a fusion protein), acetylated α-tubulin (an axoneme marker), and DAPI (a nuclear counterstain). Imaging confirmed the flagellar localization of each RFP-tagged construct, with spatial resolution of the sperm head and flagellum enabled by DAPI counterstaining. Scale bar: 20 μm. Quantification of the head-to-tail TEKT2 fluorescence intensity ratio in the indicated groups. All the data are presented as the means ± SDs. In (A-C), n = 12 biologically independent mice (A-B) or n = 12 fields from 3 biological replicates (C); the data were normalized to the mean of the control group where indicated; two-way ANOVA with Tukey's post hoc test; **** P < 0.0001. In (D), n = 3 biological replicates; proteomic data are shown as Z scores. Differential protein expression was determined by two-tailed Student's t test with FDR correction (thresholds: P < 0.05, |log₂FC| > 1). (E) shows a representative image from a single independent experiment. The TEKT2 signal was normalized to that of β-actin (whole sperm or flagella). No formal statistical testing was applied to this single replicate. In (F-G), n = 10 biologically independent mice; ≥100 sperm scored per mouse for immunofluorescence analysis; the head-to-tail TEKT2 fluorescence intensity ratio was quantified using ImageJ; unpaired two-tailed Student's t test; **** P < 0.0001.

Article Snippet: Finally, nuclear counterstaining was performed using DAPI (C1005, Beyotime) for 5 minutes.

Techniques: Analysis, Over Expression, Transmission Assay, Electron Microscopy, Expressing, Profiling, Control, Western Blot, Isolation, Immunofluorescence, Injection, Recombinant, Marker, Imaging, Construct, Fluorescence, Two Tailed Test

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: Bioengineering & Translational Medicine

Article Title: Extracellular matrix microarchitecture modulates cellular behavior and extracellular vesicle phenotypes in biomimetic tendon models

doi: 10.1002/btm2.70134

Figure Lengend Snippet: (a) Actin (green) and DAPI (blue) stain of tendon derived cells seeded on tissue culture plate (TCP) monolayer controls, healthy mimetic models and diseased mimetic models ( n = 3). Scale bar: 100 μm. (b) Nuclear aspect ratio (NAR) was quantified using ImageJ by converting microscopic image to grayscale and measuring major and minor axes of individual nuclei. A higher major‐to‐minor ratio indicates increased cellular elongations. (c) Cells on the healthy model showed greater elongation than both the monolayer control and diseased model by day 3, while the diseased model also exceeded the monolayer control. By day 5, elongation remained elevated only in the healthy model relative to the diseased, and by day 7, the healthy group exhibited significantly greater elongation than all other models. No significant differences were observed by day 10. Significance was defined as p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), p < 0.0001 (****), ns = not significant.

Article Snippet: Cellular morphology was assessed via actin staining (Invitrogen, Cat. No. A12379) and nuclear counterstaining with DAPI (Fisher Scientific, Cat. No. H‐1200‐10) ( n = 3).

Techniques: Staining, Derivative Assay, Control