Journal: Glia

Article Title: Astrocytic EAAT ‐2 Upregulation Supports Neuro‐Axonal Homeostasis in Chronic Inflammatory CNS Lesions

doi: 10.1002/glia.70243

Figure Lengend Snippet: Antibodies for immunofluorescence staining of mouse spinal cord tissue and human tissue.

Article Snippet: GFAP*** , PE , REA335|1B4 , Miltenyi Biotec , 130‐118‐351 , Human cell line , Hu, ms, rat , 1:50.

Techniques: Immunofluorescence, Staining, Tissue

Journal: Molecular Neurobiology

Article Title: Neural Expression of IL-16 Mediates Neuropathic Pain after Sciatic Injury in Mice

doi: 10.1007/s12035-026-06280-2

Figure Lengend Snippet: Expression of IL-16 in axons and Schwann cells of injured sciatic nerves. Lack of effect of neutrophil depletion on thermal hyperalgesia and IL-16 labelling in nerves. ( a1 – a4 ) Representative examples of confocal images of cross sections of injured sciatic nerves labelled with DAPI (blue, a1 ), IL-16 (green, a2 ) and the neural marker βIII-tubulin (red, a3 ). The merged image shows the colocalization of IL-16 and βIII-tubulin (yellow, a4 ), and higher-magnification images of the yellow, blue and red boxes are shown in a5 ). The yellow box shows an axon double-positive for IL-16 and βIII-tubulin (yellow). The blue box contains two small-diameter axons, one of them double-positive for IL-16 and βIII-tubulin (yellow) and the other only positive for βIII-tubulin (red). The red box indicates a βIII-tubulin-positive axon surrounded by IL-16 immunoreactivity with a shape compatible with a Schwann cell (green). ( b1 – b4 ) Confocal images corresponding to cross sections in injured sciatic nerves labelled with DAPI (blue, b1 ), IL-16 (green, b2 ) and the Schwann cell marker S100β (red, b3 ). The merged image shows the colocalization of IL-16 and S100β (yellow, b4 ). Higher-magnification images of the cell contained in the red box appear in b5 , when labelled with IL-16 (green, left hand side), S100β (red, middle image) and the merged image with DAPI (yellow, right hand side). ( c1 ) Representative merged image of IL-16, Ly6G and DAPI immunofluorescence in a longitudinal section coming from an injured nerve showing the presence of some neutrophils in a square remarked by a white dashed line. ( c2 – c5 ) Higher-magnification images of cells contained in the box, when labelled with DAPI (blue, c2 ), IL-16 (green, c3 ), Ly6G (red, c4 ) and the merged image showing the colocalization of IL-16 + and Ly6G + cells (yellow, c5 ). ( d ) Schematic representation of the experimental procedure used to evaluate the functional effect of an anti-IL-16 antibody in neutrophil-depleted mice submitted to sciatic nerve ligation 2 days before. ( e ) Decreased withdrawal latencies are shown in neutrophil-depleted mice 2 days after nerve ligation. The s.c. administration of the anti-IL-16 antibody (10 µg/kg) reversed this thermal hyperalgesia measured by the unilateral hot plate test 1 h later. Means and their corresponding standard errors are represented, •• p < 0.01 compared with the contralateral paw; ** p < 0.01 compared with values obtained in injured mice treated with IgG2bκ, two-way ANOVA followed by Tukey’s test. ( f ) Representative immunofluorescent merged image of IL-16 + traits (green) and DAPI nuclear staining (blue) in a longitudinal section of the ligated sciatic nerve from a neutrophil-depleted mouse

Article Snippet: Sections were simultaneously incubated overnight at 4 °C in a humidified chamber with the rabbit polyclonal anti-IL-16 antibody (1:100) and a rat monoclonal anti-Ly6G antibody (1:200; Bio X Cell, BE0075-1).

Techniques: Expressing, Confocal, Marker, Immunofluorescence, Functional Assay, Ligation, Hot Plate Test, Staining

Journal: Research

Article Title: Inverse Vaccine Nanomimics: In situ Antigen-Specific Immune Tolerance Induction for Durable Cardiac Repair Post-Myocardial Infarction

doi: 10.34133/research.1464

Figure Lengend Snippet: Phenotypic changes and inflammatory factor secretion of T cells stimulated by cardiac lysate antigens (CLAs). (A) Coculture protocol for dendritic cells (DCs) and T cells. The tolerogenic DCs (tDCs) present antigens to T cells; meanwhile, programmed death ligand 1 binds to programmed cell death protein 1 on T cells and inhibits T cell activation, promoting the phenotypic conversion of T cells to regulatory T cells (Tregs). Furthermore, due to the lack of activation signals such as CD80 and CD86, some T cells exhibit a weak activation state after receiving antigen presentation, and under the regulation of key factors such as interleukin-10 (IL-10) and transforming growth factor-β (TGF-β), they undergo phenotypic transition to Tregs. Under restimulation, tDCs phagocytose antigens and present them to Tregs. Tregs are rapidly activated, express high levels of Forkhead box P3 (FOXP3), and directly inhibit T cell activation, while secreting large amounts of anti-inflammatory factors. (B) Flow cytometry (FC) results for T cell phenotype. (C) Quantification of T cell phenotypes by FC. (D) T cell phenotype immunofluorescence staining results, where blue represents cell nuclei, red represents CD127, and green represents CD4. (E) Expression of interferon-γ (IFN-γ), IL-2, IL-10, and TGF-β after coculture of DCs and T cells. (F) Western blot analysis of the activation status of the mechanistic target of rapamycin (mTOR)-p70S6K-S6 pathway after treatment with Gel@PPM, Rapa, inverse vaccine nanomimics (IVNMs), and Gel@IVNMs under CLA stimulation. (G) The percentage of phosphorylation of mTOR, p70S6K, and S6 was statistically analyzed based on the gray values of the western blot results (compared to the unphosphorylated proteins). Data are presented as means ± SD, with individual points representing biological replicates. Statistical significance is indicated as ns, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: After surface staining, cells were fixed and permeabilized with Fixation/Permeabilization Concentrate and Permeabilization Buffer (E-CK-A108, Elabscience, China) according to the manufacturer’s instructions and intracellularly stained with an anti-rat FOXP3 antibody (E-AB-F1351J, Elabscience, China).

Techniques: Activation Assay, Immunopeptidomics, Flow Cytometry, Immunofluorescence, Staining, Expressing, Western Blot, Analysis, Phospho-proteomics

Journal: Research

Article Title: Inverse Vaccine Nanomimics: In situ Antigen-Specific Immune Tolerance Induction for Durable Cardiac Repair Post-Myocardial Infarction

doi: 10.34133/research.1464

Figure Lengend Snippet: Changes in dendritic cell phenotype and regulatory T cell responses in cardiac-draining lymph nodes after myocardial infarction (MI). (A) Representative flow cytometry plots showing CD11c + MHC-II + dendritic cells and programmed death ligand 1 (PD-L1) expression in the Control, Gel@PPM, Gel@PP@Rapa, Gel@OVA-IVNMs, and Gel@IVNMs groups at 7 and 56 d after MI. (B) Quantification of the proportion of PD-L1 + cells within the CD11c + MHC-II + dendritic cell population at 7 and 56 d. (C) Quantification of CD4 + CD25 + FOXP3 + regulatory T cells (Tregs) in lymph node-derived cells from the 5 treatment groups after 48 h of culture under unstimulated Control, OVA, or cardiac lysate antigen (CLA) stimulation at 7 and 56 d. (D) Representative flow cytometry plots of CD25 and Forkhead box P3 (FOXP3) expression within CD4 + T cells in the Gel@OVA-IVNMs and Gel@IVNMs groups following Control, OVA, or CLA stimulation at 7 and 56 d. Data are presented as means ± SD, with individual points representing biological replicates. Statistical significance is indicated as ns, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: After surface staining, cells were fixed and permeabilized with Fixation/Permeabilization Concentrate and Permeabilization Buffer (E-CK-A108, Elabscience, China) according to the manufacturer’s instructions and intracellularly stained with an anti-rat FOXP3 antibody (E-AB-F1351J, Elabscience, China).

Techniques: Flow Cytometry, Expressing, Control, Derivative Assay

Journal: Research

Article Title: Inverse Vaccine Nanomimics: In situ Antigen-Specific Immune Tolerance Induction for Durable Cardiac Repair Post-Myocardial Infarction

doi: 10.34133/research.1464

Figure Lengend Snippet: Phenotypic changes and inflammatory factor secretion of T cells stimulated by cardiac lysate antigens (CLAs). (A) Coculture protocol for dendritic cells (DCs) and T cells. The tolerogenic DCs (tDCs) present antigens to T cells; meanwhile, programmed death ligand 1 binds to programmed cell death protein 1 on T cells and inhibits T cell activation, promoting the phenotypic conversion of T cells to regulatory T cells (Tregs). Furthermore, due to the lack of activation signals such as CD80 and CD86, some T cells exhibit a weak activation state after receiving antigen presentation, and under the regulation of key factors such as interleukin-10 (IL-10) and transforming growth factor-β (TGF-β), they undergo phenotypic transition to Tregs. Under restimulation, tDCs phagocytose antigens and present them to Tregs. Tregs are rapidly activated, express high levels of Forkhead box P3 (FOXP3), and directly inhibit T cell activation, while secreting large amounts of anti-inflammatory factors. (B) Flow cytometry (FC) results for T cell phenotype. (C) Quantification of T cell phenotypes by FC. (D) T cell phenotype immunofluorescence staining results, where blue represents cell nuclei, red represents CD127, and green represents CD4. (E) Expression of interferon-γ (IFN-γ), IL-2, IL-10, and TGF-β after coculture of DCs and T cells. (F) Western blot analysis of the activation status of the mechanistic target of rapamycin (mTOR)-p70S6K-S6 pathway after treatment with Gel@PPM, Rapa, inverse vaccine nanomimics (IVNMs), and Gel@IVNMs under CLA stimulation. (G) The percentage of phosphorylation of mTOR, p70S6K, and S6 was statistically analyzed based on the gray values of the western blot results (compared to the unphosphorylated proteins). Data are presented as means ± SD, with individual points representing biological replicates. Statistical significance is indicated as ns, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Cells were then surface-stained with fluorophore-conjugated anti-rat CD4 (E-AB-F1105C, Elabscience, China) and CD25 (PE-30314, Finetest, China) antibodies.

Techniques: Activation Assay, Immunopeptidomics, Flow Cytometry, Immunofluorescence, Staining, Expressing, Western Blot, Analysis, Phospho-proteomics

Journal: Research

Article Title: Inverse Vaccine Nanomimics: In situ Antigen-Specific Immune Tolerance Induction for Durable Cardiac Repair Post-Myocardial Infarction

doi: 10.34133/research.1464

Figure Lengend Snippet: Changes in dendritic cell phenotype and regulatory T cell responses in cardiac-draining lymph nodes after myocardial infarction (MI). (A) Representative flow cytometry plots showing CD11c + MHC-II + dendritic cells and programmed death ligand 1 (PD-L1) expression in the Control, Gel@PPM, Gel@PP@Rapa, Gel@OVA-IVNMs, and Gel@IVNMs groups at 7 and 56 d after MI. (B) Quantification of the proportion of PD-L1 + cells within the CD11c + MHC-II + dendritic cell population at 7 and 56 d. (C) Quantification of CD4 + CD25 + FOXP3 + regulatory T cells (Tregs) in lymph node-derived cells from the 5 treatment groups after 48 h of culture under unstimulated Control, OVA, or cardiac lysate antigen (CLA) stimulation at 7 and 56 d. (D) Representative flow cytometry plots of CD25 and Forkhead box P3 (FOXP3) expression within CD4 + T cells in the Gel@OVA-IVNMs and Gel@IVNMs groups following Control, OVA, or CLA stimulation at 7 and 56 d. Data are presented as means ± SD, with individual points representing biological replicates. Statistical significance is indicated as ns, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Cells were then surface-stained with fluorophore-conjugated anti-rat CD4 (E-AB-F1105C, Elabscience, China) and CD25 (PE-30314, Finetest, China) antibodies.

Techniques: Flow Cytometry, Expressing, Control, Derivative Assay