Journal: Scientific Reports

Article Title: Intranasal delivery of nanomicelle curcumin promotes corneal epithelial wound healing in streptozotocin-induced diabetic mice

doi: 10.1038/srep29753

Figure Lengend Snippet: The effect of 7-day treatment with topical and/or intranasal nanomicelle curcumin in promoting diabetic corneal nerve regeneration after corneal epithelium abrasion was investigated. Corneas were harvested, flat-mounted, and immunostained with β-III tubulin antibody. ( A ) Images of central and peripheral corneal nerve were taken and combined as a whole flat-mounted corneal nerve. ( B ) Central and peripheral corneal nerve densities were calculated and are expressed as a percentage of that in the NC group (* P < 0.05 when compared with the DC group; ** P < 0.01, with the DC group; # P < 0.05, with the OT group; n = 3).

Article Snippet: Corneal nerve staining was performed, as described previously, with an anti-neuron-specific β-III tubulin antibody (NL1195R; R&D System Inc. Minneapolis, USA).

Techniques:

Journal: ACS nano

Article Title: Combination Nanomedicine Strategy for Preventing High-Risk Corneal Transplantation Rejection.

doi: 10.1021/acsnano.4c06595

Figure Lengend Snippet: Figure 7. Corneal nerve regeneration after HR PKP surgery. (A) Immunostaining images of corneal nerve marker β-tubulin III, scale bar 200 μm. (B) Corneal nerve regeneration was quantified as the percentage of threshold area positive for β-tubulin III staining in the center part of cornea with the diameter of 3.5 mm. Statistical analysis for B: one-way ANOVA with a Tukey post hoc test for multiple comparison. (*p ≤0.05; **p ≤0.01; and ***p ≤0.001). N = 3 for each group. All data are plotted from mean ± SEM.

Article Snippet: After that, cornea samples were washed with PBS with 0.3% Triton X-100 (Sigma-Aldrich) for 4 × 10 min followed by blocking with 2% BSA-PBS-Triton X-100 solution at room temperature for another 2 h. The cornea was incubated at 4 °C overnight in fluorescein-labeled antirat β-tubulin III antibody (R&D systems, Cat No: NL1195R).

Techniques: Immunostaining, Marker, Staining, Comparison

Journal: Journal of Neuroinflammation

Article Title: Neuronal maturation reduces the type I IFN response to orthobunyavirus infection and leads to increased apoptosis of human neurons

doi: 10.1186/s12974-019-1614-1

Figure Lengend Snippet: Committed neurons are susceptible to LACV-induced apoptosis. Representative confocal images of ( a , f , and k ) mock or ( b – e , g – j, and l – o ) 3 dpi LACV-infected COs immunohistochemically labeled with LACV (green), neuronal phenotyping antibodies (white), activated poly caspases (magenta), and nuclei (blue). Images are grouped by row using the neuronal phenotyping antibody with Sox2 being top, DCX middle, and βIII tubulin bottom. The three middle columns are images of single channels overlaid on nuclei from 3 dpi LACV-infected COs that are labeled accordingly. The far-right column ( e , j , and o ) is a combination of all four labels. The insets in e , j , and o are enlarged images of the highlighted yellow boxes in each panel. The corresponding yellow arrows in the associated individual label panels highlight the cell of interest shown in the inset. The images in the a , f , and k mock column are a combination of all four labels. All images were taken with a × 63 objective and are maximum intensity projects of 3 μm z -stacks taken with a 0.5 μm step. Scale bar in B = 20 μm and applies to all panels

Article Snippet: Antibodies used: Trustain FcXTM (Biolegend #422301, 1:1000), Sox2 (Millipore #FCMAB112, 1:50), DCX (BD Pharmigen #561505, 1:50), and βIII tubulin (R&D Systems #IC1195C, 1:50).

Techniques: Infection, Labeling

Journal: Journal of Neuroinflammation

Article Title: Neuronal maturation reduces the type I IFN response to orthobunyavirus infection and leads to increased apoptosis of human neurons

doi: 10.1186/s12974-019-1614-1

Figure Lengend Snippet: Flow cytometry analysis of neuronal cells from mock- and LACV-infected COs. Mock- ( a – d ) and LACV-infected ( e – h ) COs were non-enzymatically digested into a single cell suspension and analyzed via flow cytometry as described in the “ ” section. Two representative examples are shown. Live cells were identified and interrogated for expression of activated poly-caspases ( y -axis) and LACV expression ( x -axis). a , e Active caspase and LACV staining from the whole live cell population. Gating within the whole live cell population for Sox2 ( b , f ), DCX ( c , g ) and βIII tubulin-positive cells allowed for examination of active poly-caspase and LACV staining within each neuronal population. Proportions of LACV-infected ( i ), activated poly-caspase ( j ), and LACV-infected/activated poly-caspase double-positive ( k ) neuronal cells within mock (closed circles) or infected (open squares) COs are shown. A two-way ANOVA with a Sidak’s multiple comparisons test was used to determine significance. ** p < 0.001, **** p < 0.0001

Article Snippet: Antibodies used: Trustain FcXTM (Biolegend #422301, 1:1000), Sox2 (Millipore #FCMAB112, 1:50), DCX (BD Pharmigen #561505, 1:50), and βIII tubulin (R&D Systems #IC1195C, 1:50).

Techniques: Flow Cytometry, Infection, Suspension, Expressing, Staining

Journal: Journal of Neuroinflammation

Article Title: Neuronal maturation reduces the type I IFN response to orthobunyavirus infection and leads to increased apoptosis of human neurons

doi: 10.1186/s12974-019-1614-1

Figure Lengend Snippet: Type I IFN signaling induction in committed neurons increases cell survival. Cell viability of mock- or LACV-infected COs treated with either a IFNα2 and IFNα4 concomitantly or b IFNβ1 individually were measured using a resazurin reduction-based assay. Data is plotted as an average percent of base fluorescence measured at 590 nm for each CO. Mock IFN-treated samples are indicated by open circles, LACV vehicle-treated samples are indicated by open squares and LACV IFN-treated samples are indicated by open triangles. Fluorescence for each sample at each time point was read in triplicate at the indicated time point. A two-way ANOVA with a Sidak’s multiple comparisons test was used to determine significance. * p < 0.05. Data are representative of n = 3 mock IFNα2/4, n = 6 LACV Vehicle, n = 3 LACV IFNα2/4, n = 6 mock IFNβ1 and n = 6 LACV IFNβ1 treated COs. c Supernatants from mock (black circles), LACV IFNβ1 (green triangles), LACV IFNα2/4 (blue triangles), or LACV vehicle (red squares) treated COs were collected daily and assayed for viral RNA via qRT PCR. Data are presented as LACV RNA expression relative to an experimentally determined PFU standard. A two-way repeated measure ANOVA with a Dunnett’s multiple comparison test was performed on the antilog of the experimental values to establish differences between LACV vehicle and IFN treated samples. ** p < 0.01, * p < 0.05. *Color denotes which condition differed relative to LACV vehicle. d Quantification of immunohistochemical labeling for Sox2, DCX, and βIII tubulin in sections from the same COs shown in b are plotted as a percent positive signal of organoid area. A Kruskal-Wallis one-way ANOVA test with multiple comparisons was used to determine significance. *** p < 0.005. Representative sections of βIII tubulin staining in whole COs treated with e mock INFβ1, f LACV vehicle, or g LACV IFNβ1 are shown

Article Snippet: Antibodies used: Trustain FcXTM (Biolegend #422301, 1:1000), Sox2 (Millipore #FCMAB112, 1:50), DCX (BD Pharmigen #561505, 1:50), and βIII tubulin (R&D Systems #IC1195C, 1:50).

Techniques: Infection, Fluorescence, Quantitative RT-PCR, RNA Expression, Comparison, Immunohistochemical staining, Labeling, Staining

Journal: Current Research in Toxicology

Article Title: Neural organoids incorporating microglia to assess neuroinflammation and toxicities induced by known developmental neurotoxins ☆

doi: 10.1016/j.crtox.2025.100252

Figure Lengend Snippet: Generation and immunofluorescence characterization of planar neuroimmune organoid. Organoids are generated in 96-well plates by the addition of iPSC-derived cells to the top of a 1) PEG-based hydrogel. Neuroimmune organoids were created by sequentially adding 2) neural progenitor cells (NPCs), 3) endothelial cells (ECs) and mesenchymal cells (MSCs), and finally 4) microglia (MGs). Organoids are cultured 21–28 days from initial NPC plating prior to treatments. For the neurotoxicity assessment herein, organoids were treated on day 23 and dosing occurred for 4 days. Cell culture supernatants were collected each day. On day 4, organoids were either harvested for transcriptional analysis or fixed for immunocytochemistry analysis. Representative images show the glial components of the neuroimmune organoids. Astrocytes and microglia were stained using antibodies to detect GFAP (green, left) or IBA1 (orange, right), respectively. Neuronal staining was performed using β3 tubulin (blue, both panels). The images shown are maximum intensity projections of 20X confocal images.

Article Snippet: Donkey anti-rabbit AF555 antibody (1:500, Thermo A31572), and AF-647 conjugated anti β3 tubulin antibody (1:250, RnD Systems IC1195R), was incubated overnight at 4 °C and washed three times in PBS.

Techniques: Immunofluorescence, Generated, Derivative Assay, Cell Culture, Immunocytochemistry, Staining