mouse anti en1  (Developmental Studies Hybridoma Bank)

Journal: STAR Protocols

Article Title: A protocol for the differentiation of human embryonic stem cells into midbrain dopaminergic neurons

doi: 10.1016/j.xpro.2023.102355

Figure Lengend Snippet: Immunofluorescence of hESC-derived mDA progenitors at day 16 (A) LMX1 (floorplate marker) and FOXA2 (ventral midbrain marker), (B) LMX1 and OTX2 (forebrain and midbrain marker), (C) LMX1 and EN1 (caudal ventral midbrain marker), (D) LMX1 and NGN2 (neurogenic maker), and (E) SOX2 (neural progenitor marker) and DCX (immature neuronal marker). On day 16, most cells are LMX1 + , FOXA2 + , OTX + 2 and EN1 + mDA progenitors (A-C). Few cells expressed NGN2 (D) and DCX (E). Scale bars, 100 μm.

Article Snippet: Mouse monoclonal anti-EN1 antibody (1:50) , DSHB , Cat#4G11.

Techniques: Immunofluorescence, Derivative Assay, Marker

Journal: STAR Protocols

Article Title: A protocol for the differentiation of human embryonic stem cells into midbrain dopaminergic neurons

doi: 10.1016/j.xpro.2023.102355

Figure Lengend Snippet: Immunofluorescence of hESC-derived mDA neurons at day 56 (A–C) (A) FOXA2 and TH (DA neuron marker), (B) NURR1 (DA neuron and post-mitotic DA neuroblast marker) and TH, (C) and EN1 and TH. Scale bars, 200 μm.

Article Snippet: Mouse monoclonal anti-EN1 antibody (1:50) , DSHB , Cat#4G11.

Techniques: Immunofluorescence, Derivative Assay, Marker

Journal: STAR Protocols

Article Title: A protocol for the differentiation of human embryonic stem cells into midbrain dopaminergic neurons

doi: 10.1016/j.xpro.2023.102355

Figure Lengend Snippet:

Article Snippet: Mouse monoclonal anti-EN1 antibody (1:50) , DSHB , Cat#4G11.

Techniques: Recombinant

Journal: PLoS ONE

Article Title: Oct4-Induced Reprogramming Is Required for Adult Brain Neural Stem Cell Differentiation into Midbrain Dopaminergic Neurons

doi: 10.1371/journal.pone.0019926

Figure Lengend Snippet: ( A ) NSC-derived iPSCs were differentiated into DA neurons according to the five-stage protocol. Neural differentiation was induced by EB formation in ITSFn medium. Subsequently, the neural precursors were expanded in the presence of the growth factors FGF-2, FGF8, and Shh. Terminal differentiation was induced by growth factor withdrawal in the presence of ascorbic acid. ( B ) Graphs indicate the percentage of cells that stained positive for β-TubIII and TH, relative to nuclear Hoechst staining. Error bars indicate SEM. Three independent experiments were performed in triplicate. ( C ) Brightfield images showing EB-derived FGF2-responsive neural precursor cells. ( D ) Brightfield microphotographs of differentiated NSC-derived iPSCs, 7 days after growth factor withdrawal. ( E ) Immunofluorescence staining of neuronal cultures derived from NSC-derived iPSCs for β-TubIII (green) and TH (red). ( F ) Immunofluorescence staining for TH (red) and GABA (green), a marker typically expressed in olfactory bulb (OB) glomerular interneurons. Nuclei were counterstained with Hoechst. ( G–H ) Confocal images of neuronal cultures stained for β-TubIII (green) (G), TH (red) (G–H), En1 (blue) (G) and Pitx3 (green) (H). ( I ) Quantitative RT-PCR analysis of midbrain transcription factors, DA neurotransmission markers (TH, DAT, Pitx3, En1, Girk2, VMAT, ALDH, Nurr1) and calbindin in differentiated NSC-derived iPSCs. cDNA was isolated from differentiated adult NSC-derived iPSCs, and values were normalized to the level of β-actin. Error bars indicate SEM. Three independent experiments were performed in triplicate. Scale bar: 200 µm (C–D); 50 µm (E–F); 20 µm (G–H). ITSFn, insulin/transferrin/selenium/fibronectin.

Article Snippet: The following primary antibodies were used: sheep/rabbit anti-TH (1∶1,000; Pel-Freez Biologicals), rabbit anti-Pitx3 (1∶250, Zymed), mouse anti-En1 (1∶50, DSHB), goat anti-Foxa2 (1∶100, Santa Cruz), rabbit/chicken anti β-TubIII (Tuj1, Covance), rabbit anti-GFAP (1∶1000, Dako), mouse anti-SSEA-1 (0.4 µg/ml; DSHB), mouse anti Oct4 (1∶100, Santa Cruz Biotechnology), rabbit anti Nanog (1∶100, Bethyl), mouse anti NeuN (1∶1000, Chemicon), mouse anti SSEA1 (1∶50, Chemicon), rabbit anti GAD67 (1∶5000, Sigma), rabbit anti GABA (1∶5000, Sigma), mouse anti AFP (1;100, R&D Systems), goat anti brachyury (1∶100, Santa Cruz Biotechnology).

Techniques: Derivative Assay, Staining, Immunofluorescence, Marker, Quantitative RT-PCR, Isolation

Journal: STAR Protocols

Article Title: An Optimized Protocol for the Generation of Midbrain Dopamine Neurons under Defined Conditions

doi: 10.1016/j.xpro.2020.100065

Figure Lengend Snippet:

Article Snippet: Mouse monoclonal anti-EN1 , DSHB , 4G11, RRID: AB_528219.

Techniques: Recombinant, Knock-Out