Journal: Cell Reports Methods

Article Title: Methods for culturing adult CNS neurons reveal a CNS conditioning effect

doi: 10.1016/j.crmeth.2022.100255

Figure Lengend Snippet:

Article Snippet: Biotin-Antibody Cocktail , Miltenyi , 130-115-390.

Techniques: Control, Recombinant, Sterility, Flow Cytometry, Isolation, Software

Journal: Neurology Research International

Article Title: Subarachnoid Transplant of the Human Neuronal hNT2.19 Serotonergic Cell Line Attenuates Behavioral Hypersensitivity without Affecting Motor Dysfunction after Severe Contusive Spinal Cord Injury

doi: 10.1155/2011/891605

Figure Lengend Snippet: Neural and human markers with differentiation of the hNT2.19 cell line in vitro. The hNT2.19 cell line was treated for two weeks with retinoic acid and mitotic inhibitors and lifted to substrate-coated 8-well plastic TC slides for differentiation and immunohistochemistry for neuron-specific markers. As soon as 4 days in vitro, a variety of neural markers appeared, which remained strong until at least 6 wks of differentiation: TuJ1 (a), hNSE (b), NFL (c), NFM (d), and NFH (e). For comparison, the negative control hNT2.6 cell line was cultured similarly as the hNT2.19 cells and is here stained for TuJ1 (f). Magnification bar = 20 nm, (a–f).

Article Snippet: For immunohistochemistry of sectioned spinal cord tissues, the polyclonal antibody anti-5HT (ab10385; dilution 1/100 (in vivo)) was purchased from Abcam Inc, Cambridge, MA, and the antihuman TuJ1 antibody (Neuron-specific class III beta-tubulin) was purchased from Neuromics, Edina, MN (MO15013; dilution 1/100 (in vivo).

Techniques: In Vitro, Immunohistochemistry, Comparison, Negative Control, Cell Culture, Staining

Journal: Neurology Research International

Article Title: Subarachnoid Transplant of the Human Neuronal hNT2.19 Serotonergic Cell Line Attenuates Behavioral Hypersensitivity without Affecting Motor Dysfunction after Severe Contusive Spinal Cord Injury

doi: 10.1155/2011/891605

Figure Lengend Snippet: Transplant of hNT2.19 and hNT2.6 cell lines in the severe contusive SCI model: TuJ1 and 5HT immunohistochemistry. Rats were injured with severe contusive SCI followed at two weeks by hNT2.6 (a, b) or hNT2.19 (c, d) cell grafts. Sagittal spinal cord sections were examined at 8 wks after SCI for evidence of surviving lumbar subarachnoid hNT2.6 (a, b) or hNT2.19 (c, d) cell line grafts, utilizing TuJ1 (a, c) or 5HT (b, d) immunohistochemistry. The hNT2.19 and control hNT2.6 (10 6 cells/injection), which had been differentiated for two weeks in vitro, were injected into the subarachnoid space two weeks after the SCI. Cell graft sites were colocalized with 5HT (b, d) and the human-specific marker TUJ1 (neuron-specific class III β -tubulin; (a, c)). There are many surviving hNT2.19 (c) and hNT2.6 (a) grafted cells visible on the pial surface, which stain for TuJ1 (arrows) at the end of the experiment, 56 days after SCI and about 6 weeks after cell transplant. Adjacent sections with the same grafted hNT2.19 (d) and hNT2.6 cells (b) are stained for 5HT, but only the hNT2.19 cells (d) are labeled for 5HT (arrows).

Article Snippet: For immunohistochemistry of sectioned spinal cord tissues, the polyclonal antibody anti-5HT (ab10385; dilution 1/100 (in vivo)) was purchased from Abcam Inc, Cambridge, MA, and the antihuman TuJ1 antibody (Neuron-specific class III beta-tubulin) was purchased from Neuromics, Edina, MN (MO15013; dilution 1/100 (in vivo).

Techniques: Immunohistochemistry, Control, Injection, In Vitro, Marker, Staining, Labeling

Journal: The American Journal of Pathology

Article Title: Antibiotic-Induced Dysbiosis of Gut Microbiota Impairs Corneal Nerve Regeneration by Affecting CCR2-Negative Macrophage Distribution

doi: 10.1016/j.ajpath.2018.08.009

Figure Lengend Snippet: Alteration of corneal homeostasis in mice after antibiotic (Abx) treatment in drinking water. A: RNA sequencing was used to detect changes in gene expression in the corneas from Abx-treated mice compared with those from the control mice (treated with sterile water). Biological processes were assigned to genes by using annotated gene ontology (GO). The bar of the color represents the aggregation degree of genes in one kind of biological process. B: Corneal whole-mount immunostaining of anti–β-III tubulin NL557. The cornea with complete limbus can be divided into five ( 1–5 ) zones from limbus to central cornea under the microscope by using a 40× objective oil immersion lens. Left panel: Zone 1 is in the limbus, and zones 2–5 are in the cornea. Right five panels: Enlarged views in the five zones in one direction. C: Definition of nerve fibers located in different corneal layers. D–F: Comparison of corneal nerve density in stratified epithelium, subbasal layer, and stroma between Abx-treated and control mice (sterile water treated). Left panels: Nerve density in each corneal zone from control and Abx-treated mice. Right panels: Total nerve density from the second to fifth zones in the cornea of control and Abx-treated mice. Data are expressed as means ± SD ( D–F ). n = 2 independent experiments (5 mice per experiment; A ); n = 6 mice in each group ( D–F ). ∗ P < 0.05. Scale bars: 200 μm ( B , left panel ); 25 μm ( B , right five panels , and C ).

Article Snippet: The treated corneas were then incubated with anti–β-III tubulin conjugated with NL557 (number NL1195R; R&D Systems) (1:10) or anti-mouse CD64 fluorescein isothiocyanate (number 139315; BioLegend, San Diego, CA) (1:100) antibodies at 4°C overnight.

Techniques: RNA Sequencing, Gene Expression, Control, Sterility, Immunostaining, Microscopy, Comparison

Journal: The American Journal of Pathology

Article Title: Antibiotic-Induced Dysbiosis of Gut Microbiota Impairs Corneal Nerve Regeneration by Affecting CCR2-Negative Macrophage Distribution

doi: 10.1016/j.ajpath.2018.08.009

Figure Lengend Snippet: Effect of macrophage (MФ) depletion on corneal nerve regeneration. A: Anti–β-III tubulin NL557 and CD64 fluorescein isothiocyanate antibody costaining of cornea. B: RT-PCR evaluation of flow cytometry–sorted CCR2 − and CCR2 + corneal macrophages for the expression of neurotrophin genes. C and E: The change of neurotrophin gene expression in corneal tissues at 8 days after epithelial abrasion. CCR2 − corneal macrophages were depleted by subconjunctival injection of anti–colony stimulating factor 1 receptor (CSF1R) antibody, and CCR2 + corneal macrophages were depleted by i.p. injection of CCR2 antagonist BMS CCR2 22. D and F: Change in the density of subbasal nerves at 8 days after epithelial abrasion in mice depleted of CCR2 − or CCR2 + macrophages. G and H: The change in corneal sensitivity after corneal epithelial abrasion in mice depleted of CCR2 − or CCR2 + macrophages. Data are expressed as means ± SD. n = 3 independent experiments [10 mice per experiment ( B ) and 6 mice per experiment ( C and E )]; n = 6 mice in each group ( D and F–H ). ∗ P < 0.05, ∗∗ P < 0.01. Scale bar = 100 μm ( A ).

Article Snippet: The treated corneas were then incubated with anti–β-III tubulin conjugated with NL557 (number NL1195R; R&D Systems) (1:10) or anti-mouse CD64 fluorescein isothiocyanate (number 139315; BioLegend, San Diego, CA) (1:100) antibodies at 4°C overnight.

Techniques: Reverse Transcription Polymerase Chain Reaction, Flow Cytometry, Expressing, Gene Expression, Injection