rabbit antisynapsin1 d12g5 xp monoclonal antibody  (Cell Signaling Technology Inc)

Journal: Cell Transplantation

Article Title: Neuronal Cell Sheets of Cortical Motor Neuron Phenotype Derived from Human iPSCs

doi: 10.1177/0963689717720280

Figure Lengend Snippet: Immunohistochemical analyses of mouse brains grafted with human induced pluripotent stem cells–derived neuronal cell sheet of cortical motor neuron phenotype. The expressions of neuron-associated proteins in the brain sections were analyzed 21 d after the transplantation (at day 42 in ). (A1–A4) Anti-human NCAM staining (green) and antisynapsin1 staining (red). Human NCAM+ cells showed expression of synapsin1, suggesting neural connections between human neurons and mouse neurons. Marginal detachment of the grafted sheet had occurred during staining procedure. (B1–B4) Anti-CTIP2 staining (green) and anti-hNuc staining (red). CTIP2-positive human neurons moved to and resided in the damaged cortex. CTIP2-positive human neurons located underneath the CTIP2-negative human neurons. (B5) Higher magnification of central area of panel B4. (C1–C4) Anti-Foxp2 staining (green) and anti-hNuc staining (red). Foxp2-positive human neurons located in the damaged cortex. (C5) Higher magnification of central area of panel C4. (D1–D4) Anti-Fezf2 staining (green) and anti-hNuc staining (red). (D5) Higher magnification of central area of panel D4. Fezf2-positive human neurons made cell layer underneath the Fezf2-negative human neurons, resembling layered structure of motoneurons. (D6) H&E staining of the same cell sheet grafted on the damaged motor cortex. (E1–E4) Anti-CTIP2 staining (green) and anti-Foxp2 staining (red). Majority of CTIP2-positive human neurons in the damaged cortex coexpressed Foxp2. The CTIP2 and Foxp2 double-positive neurons existed underneath the double negative (nonmotoneuron phenotype) 4′,6-diamidino-2-phenylindole (DAPI)-positive neurons. (F1–F4) Anti-neurofilament meddle chain (NFM, green) and anti-hNuc staining (red). (G1–G4) Anti-NFM (green) and anti-hNuc staining (red) in the brain of single-cell suspension transplanted mouse. (H1– H4) Anti-NFM (green) and anti-hNuc staining (red) in the brain of PBS-injected mouse. Panels of the left vertical row are a schematic representation of each DAPI staining. A white horizontal bar in panel A4 represents 50 μm in panels A1–A4, B1–B4, and D1–D4, F1–F4, G1–G4, H1–H4; 25 μm in panels B5, C1–C4, D5, and E1–E4; and 12.5 μm in panel C5. CTIP2, COUP-TF-interacting protein 2; Foxp2, forkhead box p2; Fezf2, forebrain embryonic zinc finger family zinc finger 2; hNuc, human nuclei; H&E, hematoxylin and eosin; PBS, phosphate-buffered saline.

Article Snippet: Immunofluorescence staining was conducted as reported previously., , , , The following antibodies were used: rabbit anti-neurofilament middle chain (Millipore, Billerica, MA, USA), mouse anti-βIII tubulin (Promega, Madison, WI, USA), rabbit anti-CRIM1 (Sigma-Aldrich), rat anti-CTIP2 (Abcam, Cambridge, MA, USA), anti-Fezf2 (Abcam), rabbit anti-Foxp2 (Abcam), mouse anti-human Neural cell adhesion molecule (NCAM) (hNCAM; Santa Cruz Biotechnology, Dallas, TX, USA), antisynapsin1 (Millipore), rabbit anti-Nanog (ReproCELL, Kanagawa, Japan), mouse anti-Oct3/4 (Santa Cruz Biotechnology), rabbit anti-Pax6 (BioLegend, San Diego, CA, USA), and mouse anti-human nuclei (Abnova, Taipei City, Taiwan).

Techniques: Immunohistochemical staining, Derivative Assay, Transplantation Assay, Staining, Expressing, Injection