Journal: PLoS ONE

Article Title: Bilateral Nerve Alterations in a Unilateral Experimental Neurotrophic Keratopathy Model: A Lateral Conjunctival Approach for Trigeminal Axotomy

doi: 10.1371/journal.pone.0070908

Figure Lengend Snippet: Representative histological photographs of corneal nerves stained with anti-βIII tubulin FITC-conjugated antibody. Normal subbasal (A) and stromal (E) nerve plexus. Subbasal nerve plexus completely disappeared (B; day 1, C; day 7, D; day 14) and stromal nerve definitely decreased from day1 (F; day 1, G; day 7, H; day 14) and after trigeminal axotomy. The nerve of the contralateral eye decreased from day 1 (J; day 1, K; day 7, L; day 14).

Article Snippet: Corneas were then stained with monoclonal NL637-conjugated anti-β-III tubulin antibody (anti-Neuron-specific β-III Tubulin-NL637, R&D systems Inc. Minneapolis, MN; dilution of 1∶100) at 4C degree overnight.

Techniques: Staining

Journal: PLoS ONE

Article Title: Bilateral Nerve Alterations in a Unilateral Experimental Neurotrophic Keratopathy Model: A Lateral Conjunctival Approach for Trigeminal Axotomy

doi: 10.1371/journal.pone.0070908

Figure Lengend Snippet: Representative histological photographs of corneal nerves stained with anti-βIII tubulin FITC-conjugated antibody. Normal subbasal (A) and stromal (E) nerve plexus.Subbasal nerve plexus completely disappeared (B; day 1, C; day 7, D; day 14) (A) and stromal nerve definitely decreased (B) from day 1 (F; day 1, G; day 7, H; day 14) after trigeminal axotomy. The nerve of the contralateral eye did not change (J; day 1, K; day 7, L; day 14).

Article Snippet: Corneas were then stained with monoclonal NL637-conjugated anti-β-III tubulin antibody (anti-Neuron-specific β-III Tubulin-NL637, R&D systems Inc. Minneapolis, MN; dilution of 1∶100) at 4C degree overnight.

Techniques: Staining

Journal: Cell Death Discovery

Article Title: PGC-1a mediated mitochondrial biogenesis promotes recovery and survival of neuronal cells from cellular degeneration

doi: 10.1038/s41420-024-01953-0

Figure Lengend Snippet: A Representative bright field (BF) and immunofluorescence images of 5% EtOH treatment (vol/vol, 1 and 3 h) induced neurite retraction, and its regeneration after washing EtOH (Washed, 4 and 20 h) in neuronal PC12 cells. Green: neuron-specific β-III tubulin; Blue: DAPI. Scale bar: 50 µm. B , C Quantification results of mean neurite length per cell and the percentage of cells with neurites. *** P < 0.001, vs. control group; ## P < 0.01, ### P < 0.001, vs. EtOH 3 h group. EtOH ethanol.

Article Snippet: For immunostaining, cells were incubated with primary antibodies against neuron-specific β-III tubulin (MAB1195, R&D systems, USA), LC3B (NB100-2220, Novus Biologicals, USA), TOM20 (42406S, Cell Signaling Technology), and Drp1 (8570S, Cell Signaling Technology) overnight at 4 °C.

Techniques: Immunofluorescence, Control

Journal: The Journal of Immunology Author Choice

Article Title: CD36-Mediated Hematoma Absorption following Intracerebral Hemorrhage: Negative Regulation by TLR4 Signaling

doi: 10.4049/jimmunol.1400054

Figure Lengend Snippet: Enhanced CD36 expression in perihematomas following ICH. (A) Detection of CD36 mRNA expression in mice subjected to ICH using real-time quantitative RT-PCR. The data are expressed as fold increases relative to naive animals. **p < 0.01 versus sham, n = 6. (B) Detection of CD36 protein expression in mice subjected to ICH using Western blot. **p < 0.01 versus sham, n = 6. (C) Detection of CD36 expression in astrocytes, neurons, and microglia in the perihematomal tissues of mice using flow cytometry at 3 d after ICH. The left panel is the representative flow cytometry plot and the lower right panel is the percentage of CD36+ cells in neurons, astrocytes, and microglia. Microglia were marked by CD45intCD11b+, whereas neurons were identified as β-III tubulin+, and astrocytes were identified as GFAP+. **p < 0.01 versus sham, ##p < 0.01 versus astrocytes or neurons, n = 3. (D) Detection of CD36 expression in human perihematomal tissues of ICH patients using fluorescence immunohistochemistry; CD36 expression was labeled with a CD36 Ab (red), astrocytes, neurons, and microglia were labeled with GFAP (green), Neun (green), and Iba-1 (green), respectively. The merged images of the overlay of CD36 together with astrocytes, neurons, and microglia were shown as yellow, and the nuclei were stained with DAPI (blue). The arrows indicate positive cells. Scale bars, 80 μm.

Article Snippet: The cells were then incubated with anti-mouse GFAP–Fluor 660 (1:200; eBioscience) and anti-mouse neuron-specific β-III tubulin PerCP (1:200; R&D Systems, Bingdon, U.K.) for 30 min at 4°C.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Flow Cytometry, Fluorescence, Immunohistochemistry, Labeling, Staining

Journal: iScience

Article Title: Preclinical validation of human recombinant glutamate-oxaloacetate transaminase for the treatment of acute ischemic stroke

doi: 10.1016/j.isci.2024.111108

Figure Lengend Snippet:

Article Snippet: Neuron-specific beta-III tubulin APC-conjugated antibody, R&D Systems , #IC1195A.

Techniques: Plasmid Preparation, Recombinant, Pore Size, Lysis, Colorimetric Assay, Caspase-3 Assay, Activity Assay, Glutamate Assay, Membrane, DC Protein Assay, Cell Culture, Software, Fluorescence, Sampling, Mass Spectrometry, Magnetic Resonance Imaging, Confocal Microscopy, Flow Cytometry, High Performance Liquid Chromatography

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