paraformaldehyde solution  (Thermo Fisher)

Journal: Bioactive Materials

Article Title: Inhalable PD-L1-engineered hybrid cellular vesicles suppress excessive neutrophil activation and restore mitochondrial homeostasis to alleviate ischemia–reperfusion lung injury and pneumonia

doi: 10.1016/j.bioactmat.2026.03.024

Figure Lengend Snippet: Characterization of Res-PD-L1@nmEVs . (A) Schematic illustration of the Res-PD-L1@nmEVs synthesis procedure. (B-D) Representative transmission electron microscopy (TEM) images, dynamic light scattering (DLS) size distributions, and zeta potential measurements of nEVs, PD-L1@mEVs, PD-L1@nmEVs, and Res-PD-L1@nmEVs. (E) PD-L1 expression in PD-L1-overexpressing MSCs (OE-PD-L1) and negative control (NC) MSCs, and CD11b expression in HL60 cells before and after DMSO stimulation, as determined by Western blot. (F) Expression levels of neutrophil membrane markers (CD11b, CXCR2, RAGE, TLR2) and the exosomal marker CD63 in the four EV types. (G) Fluorescence co-localization images of DiO-labeled nEVs (green) and DiL-labeled PD-L1@mEVs (red) after fusion, demonstrating hybrid vesicle formation. (H) Size stability of Res-PD-L1@nmEVs stored at 4 °C and 37 °C for 7 days. (I-K) Binding and neutralization capacity of Res-PD-L1@nmEVs against inflammatory cytokines (TNF-α, IL-6, IL-1β) in vitro. ∗ vs. 0ug/ml; # vs. 100 μg/ml, p < 0.05, n = 5.

Article Snippet: Immediately after euthanasia, lung tissues were harvested and fixed with electron microscopy fixative (G1102, Servicebio), followed by post-fixation in 1% osmium tetroxide for 2 h. The specimens were then dehydrated through a graded ethanol series, infiltrated with epoxy resin, and embedded.

Techniques: Transmission Assay, Electron Microscopy, Zeta Potential Analyzer, Expressing, Negative Control, Western Blot, Membrane, Marker, Fluorescence, Labeling, Binding Assay, Neutralization, In Vitro

Journal: Bioactive Materials

Article Title: Inhalable PD-L1-engineered hybrid cellular vesicles suppress excessive neutrophil activation and restore mitochondrial homeostasis to alleviate ischemia–reperfusion lung injury and pneumonia

doi: 10.1016/j.bioactmat.2026.03.024

Figure Lengend Snippet: Res-PD-L1@nmEVs Attenuate Inflammation and Oxidative Damage in Lung Epithelial Cells In Vitro . (A-B) Flow cytometric analysis and quantification (B) of DiO-labeled Res-PD-L1@nmEVs uptake by BEAS-2B cells under H/R conditions after pretreatment with different endocytic inhibitors (chlorpromazine, chloroquine, and filipin) or incubation at 4 °C. (C) mRNA expression levels of IL-6, TNF-α, and IL-1β in BEAS-2B cells with or without H/R injury following pretreatment with Res, nEVs, PD-L1@mEVs, PD-L1@nmEVs, or Res-PD-L1@nmEVs. (D-E) Representative fluorescence images (D) and quantitative analysis (E) of cell proliferation assessed by BrdU incorporation (red; nuclei stained with DAPI, blue). Scale bar: 50 μm. (F-G) Apoptosis rates detected by flow cytometry (F) and flow cytometric analysis of Annexin V-positive BEAS-2B cells under the indicated conditions (G). (H–K) Fluorescence microscopy images and quantitative analysis of intracellular nitric oxide (NO, green) (H-I) and reactive oxygen species (ROS, red) (J-K). Scale bar: 100 μm. (L) Flow cytometry analysis of intracellular ROS levels. (M − O) Levels of malondialdehyde (MDA) (M), superoxide dismutase 2 (SOD2) activity (N), and glutathione (GSH) content (O) in cells. (P-Q) Cell migration ability evaluated by wound healing assay under different treatments. ∗ vs. Control; # vs. H/R; & vs. H/R + PD-L1@nmEVs, p < 0.05.

Article Snippet: Immediately after euthanasia, lung tissues were harvested and fixed with electron microscopy fixative (G1102, Servicebio), followed by post-fixation in 1% osmium tetroxide for 2 h. The specimens were then dehydrated through a graded ethanol series, infiltrated with epoxy resin, and embedded.

Techniques: In Vitro, Labeling, Incubation, Expressing, Fluorescence, BrdU Incorporation Assay, Staining, Flow Cytometry, Microscopy, Activity Assay, Migration, Wound Healing Assay, Control

Journal: Bioactive Materials

Article Title: Inhalable PD-L1-engineered hybrid cellular vesicles suppress excessive neutrophil activation and restore mitochondrial homeostasis to alleviate ischemia–reperfusion lung injury and pneumonia

doi: 10.1016/j.bioactmat.2026.03.024

Figure Lengend Snippet: Res-PD-L1@nmEVs Restores Mitochondrial Homeostasis and Improves Energy Metabolism BEAS-2B cells were pretreated with Res, nEVs, PD-L1@mEVs, PD-L1@nmEVs, or Res-PD-L1@nmEVs followed by H/R stimulation for subsequent analysis. (A) Representative immunofluorescence images showing the expression and localization of PINK1 (green) and the mitochondrial marker TOMM20 (red), indicating activation of mitophagy. Nuclei were stained with DAPI (blue). Scale bar: 50 μm. (B) Quantitative analysis of PINK1 fluorescence intensity. (C) Expression and localization of autophagy-related proteins LC3B and Beclin-1 detected by immunofluorescence. (D-E) Quantitative analysis of LC3B (D) and Beclin-1 (E) fluorescence intensity. (F) Mitochondrial membrane potential assessed by JC-1 staining and flow cytometry. (G) Oxygen consumption rate (OCR) profiles of lung epithelial cells under different treatments. (H-K) Key mitochondrial respiration parameters: basal respiration (H), maximal respiration (I), proton leak (J), and ATP production (K). (L) Representative confocal microscopy images of mitochondria stained with MitoTracker (green) and lysosomes stained with LysoTracker (red), demonstrating mitochondrial-lysosomal colocalization. Scale bar: 5 μm ∗ vs. Control; # vs. H/R; & vs. H/R + PD-L1@nmEVs, p < 0.05.

Article Snippet: Immediately after euthanasia, lung tissues were harvested and fixed with electron microscopy fixative (G1102, Servicebio), followed by post-fixation in 1% osmium tetroxide for 2 h. The specimens were then dehydrated through a graded ethanol series, infiltrated with epoxy resin, and embedded.

Techniques: Immunofluorescence, Expressing, Marker, Activation Assay, Staining, Fluorescence, Membrane, Flow Cytometry, Confocal Microscopy, Control

Journal: Bioactive Materials

Article Title: Inhalable PD-L1-engineered hybrid cellular vesicles suppress excessive neutrophil activation and restore mitochondrial homeostasis to alleviate ischemia–reperfusion lung injury and pneumonia

doi: 10.1016/j.bioactmat.2026.03.024

Figure Lengend Snippet: Res-PD-L1@nmEVs Suppresses Neutrophil Activation and Preserves Mitochondrial Integrity via PD-L1 Delivery (A-B) Rats subjected to lung IRI received nebulized administration of different formulations (Res, nEVs, PD-L1@mEVs, PD-L1@nmEVs, or Res-PD-L1@nmEVs) before ischemia and after reperfusion. Lung tissues were collected 2 h post-reperfusion. (A) Representative immunofluorescence images showing the expression and localization of CD11b (green), MPO (red), and PD-1 (yellow) in lung sections across treatment groups. (B) Enlarged view of the IRI group from (A). (C-D) mRNA levels of CD95 (C) and CD206 (D) in lung tissues. (E-F) Levels of myeloperoxidase (MPO) (E) and matrix metalloproteinase-9 (MMP-9) (F) in bronchoalveolar lavage fluid (BALF). (G-I) (G) Representative transmission electron microscopy (TEM) images of lung tissues (scale bar: 2 μm). (H) Proportion of damaged mitochondria. (I) Average number of mitophagic events per cell. (J) Immunofluorescence co-localization of mitochondrial marker TOMM20 (red) and EpCAM (green) in lung tissues (nuclei stained with DAPI, scale bar: 50 μm). (K-L) Protein expression levels of Beclin-1 (K) and LC3 (L) in lung tissues, with insets showing immunofluorescence co-localization of Beclin-1 (green) and LC3 (red) across treatment groups (nuclei stained with DAPI, scale bar: 50 μm). ∗ vs. Sham; # vs. IRI; & vs. IRI + PD-L1@nmEVs, p < 0.05.

Article Snippet: Immediately after euthanasia, lung tissues were harvested and fixed with electron microscopy fixative (G1102, Servicebio), followed by post-fixation in 1% osmium tetroxide for 2 h. The specimens were then dehydrated through a graded ethanol series, infiltrated with epoxy resin, and embedded.

Techniques: Activation Assay, Immunofluorescence, Expressing, Transmission Assay, Electron Microscopy, Marker, Staining

Journal: iScience

Article Title: Targeting Rap1-YAP1 mechanosignaling for ameliorating acute IOP elevation-induced trabecular meshwork dysfunction

doi: 10.1016/j.isci.2026.116268

Figure Lengend Snippet: Structural evaluation of the mouse TM following acute IOP elevation (A) Photographs reveal that mice underwent direct cannulation of the anterior chamber (AC) in the eye. Eyeballs were harvested immediately after the procedure, and the gross appearance is illustrated in the right. (B) Intraocular pressure (IOP) measurement following acute ocular hypertension. IOP was measured at baseline (0 days), 3 days, and 7 days following 45 mmHg pressure for 1 h. Data represent mean ± SD. two-way ANOVA was used for comparing IOP between control and HIOP groups across time points. Post hoc test: ns, not significant, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 vs. control group at corresponding time point. N = 9 mice per group. (C) Representative hematoxylin staining of anterior chamber angles 3 days after IOP elevation. Magnification bars: 1 mm. (D) Ultrastructural appearance of the TM tissues by transmission electron microscopy. Leftmost: representative lower magnification image of the TM and SC. Right: higher magnification of the TM cells. The regions encased in black boxes are magnified. TM, trabecular meshwork; SC, Schlemm’s canal; red arrow indicates giant vacuoles.

Article Snippet: Following dissection, the TM or HTM cells grown on coverslips were immediately immersed in fresh transmission electron microscopy fixative (G1102; Servicebio, Wuhan, China) for 48 h. The samples then underwent post-fixation in a 1% OsO 4 aqueous solution for 2 h at room temperature.

Techniques: Control, Staining, Transmission Assay, Electron Microscopy

Journal: iScience

Article Title: Targeting Rap1-YAP1 mechanosignaling for ameliorating acute IOP elevation-induced trabecular meshwork dysfunction

doi: 10.1016/j.isci.2026.116268

Figure Lengend Snippet: Pathological changes in HTM cells under high hydrostatic pressure (A and B) Representative examples of human trabecular meshwork (HTM) cells cultured under control (con) or high hydrostatic pressure for 12 h, 1 day (1 D), and 3 days (3 D) immunolabeled for EDU (A) and TUNEL (B) assay. Scale bars, 50 μm. Bar graphs summarizing the effects of high hydrostatic pressure on the proliferation and death of HTM cells. N = 3. Magnification bars, 100 μm. (C) Histological evaluation of different groups of HTM cells by transmission electron microscopy. N = 3. Magnification bars, 50, 10, and 2 μm from the left to the right. (D) Representative fluorescence micrographs of F-actin in HTMs subjected to 12 h, 1D, and 3D culture under high hydrostatic pressure and the control, with nuclei and F-actin labeling shown in blue and green, respectively. The asterisk indicates cross-linked actin network (CLAN+) cells and the percentage of CLAN+ cells was analyzed. N = 5. Magnification bars, 25 μm. (E) Expression of fibronectin, collagen I and α-SMA in the different groups of HTM cells by western blots. N = 6. ns, not significant, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. Data represent mean ± SD.

Article Snippet: Following dissection, the TM or HTM cells grown on coverslips were immediately immersed in fresh transmission electron microscopy fixative (G1102; Servicebio, Wuhan, China) for 48 h. The samples then underwent post-fixation in a 1% OsO 4 aqueous solution for 2 h at room temperature.

Techniques: Cell Culture, Control, Immunolabeling, TUNEL Assay, Transmission Assay, Electron Microscopy, Fluorescence, Labeling, Expressing, Western Blot