- 14-9843-82 - Invitrogen Antibodies NES antibody

Submitted references Pannexin 1 Influences Lineage Specification of Human iPSCs.

Noort RJ, Christopher GA, Esseltine JL
Frontiers in cell and developmental biology 2021;9:659397

Initiation of Pancreatic Cancer: The Interplay of Hyperglycemia and Macrophages Promotes the Acquisition of Malignancy-Associated Properties in Pancreatic Ductal Epithelial Cells.

Otto L, Rahn S, Daunke T, Walter F, Winter E, Möller JL, Rose-John S, Wesch D, Schäfer H, Sebens S
International journal of molecular sciences 2021 May 11;22(10)

Connexin 43 Gene Ablation Does Not Alter Human Pluripotent Stem Cell Germ Lineage Specification.

Christopher GA, Noort RJ, Esseltine JL
Biomolecules 2021 Dec 22;12(1)

Molecular analyses of glioblastoma stem-like cells and glioblastoma tissue.

Wallenborn M, Xu LX, Kirsten H, Rohani L, Rudolf D, Ahnert P, Schmidt C, Schulz RM, Richter M, Krupp W, Mueller W, Johnson AA, Meixensberger J, Holland H
PloS one 2020;15(7):e0234986

Herpes simplex virus type 1 infection leads to neurodevelopmental disorder-associated neuropathological changes.

Qiao H, Guo M, Shang J, Zhao W, Wang Z, Liu N, Li B, Zhou Y, Wu Y, Chen P
PLoS pathogens 2020 Oct;16(10):e1008899

Protective effects of primary neural stem cell treatment in ischemic stroke models.

Yu X, Wang X, Zeng S, Tuo X
Experimental and therapeutic medicine 2018 Sep;16(3):2219-2228

Sphere-Derived Multipotent Progenitor Cells Obtained From Human Oral Mucosa Are Enriched in Neural Crest Cells.

Abe S, Yamaguchi S, Sato Y, Harada K
Stem cells translational medicine 2016 Jan;5(1):117-28

Histone modification profiling reveals differential signatures associated with human embryonic stem cell self-renewal and differentiation.

Bhanu NV, Sidoli S, Garcia BA
Proteomics 2016 Feb;16(3):448-58

Functional differentiation of human brain progenitor cells.

Messam CA, Ding S, Haydon PG
Neuron glia biology 2006 Aug;2(3):187-98

Redox-mediated enrichment of self-renewing adult human pancreatic cells that possess endocrine differentiation potential.

Linning KD, Tai MH, Madhukar BV, Chang CC, Reed DN Jr, Ferber S, Trosko JE, Olson LK
Pancreas 2004 Oct;29(3):e64-76

Coexpression of nestin in neural and glial cells in the developing human CNS defined by a human-specific anti-nestin antibody.

Messam CA, Hou J, Major EO
Experimental neurology 2000 Feb;161(2):585-96

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: U251 cell lysates prepared under non-reducing (lane 1) or reducing (lane 2) conditions were resolved by SDS-PAGE then immunoblotted with 5 µg/mL Anti-Human Nestin Purified.Bands were visualized using Anti-Mouse IgG HRP.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-Nestin Monoclonal Antibody (10C2), eBioscience™ (Product # 14-9843-82) and a 200 kDa band corresponding to Nestin was observed in NSCs and NSCs differentiated to neurons. Whole cell extracts (30 µg lysate) of iPSC (Lane 1), NSC (Lane 2), NSC differentiated to neurons (Lane 3) and Jurkat (Lane 4) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0321BOX). Resolved proteins were then transferred onto a Nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1 µg/mL) and detected by chemiluminescence with Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunocytochemistry of fixed and permeabilized SKNSH cells using 5 µg/mL Anti-Human Nestin Purified, followed by 10 µg/mL F (ab')2 Anti-Mouse IgG eFluor® 570.Nuclei are stained with DAPI.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of Nestin was performed using 70% confluent log phase NSC cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with Nestin Monoclonal Antibody (10C2), eBioscience™ (Product # 14-9843-82) at 5 µg/mL in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32766, 1:2000 dilution), for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b: Blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300 dilution). Panel d represents the merged image showing cytoplasmic localization. Panel e represents iPSC cells having no expression of Nestin. Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Supportive validation

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Experimental details: Fig 1 HSV-1-infected NSCs exhibited increased cell apoptosis. (A) Schematic representation of the experimental pipeline used in the differentiation process of hiPSC-derived neurons. (B) Confirmation of the characteristic expression of hiPSCs, hiPSC-derived NSCs, and NSC-derived neurons. Immunolabeling of SOX2 and Nestin in hiPSC-derived NSCs at the day 7; NSCs were further differentiated into neurons illustrated using MAP2 and NeuN immunofluorescence after the day 21 of neural induction. Scale bars represent 50 mum. (C) Sample images of hiPSCs, NSCs and neurons 24 hours after infection with HSV-1, immunostained for HSV1 gE envelop protein (green) and DAPI (blue). The images were taken using an Olympus microscope. Scale bars represent 100 mum. (D) Quantification of infection efficiency for different cell types, including hiPSCs, hiPSC-derived NSCs, and NSC-derived neurons. Data represent the mean +- SEM. *p < 0.05 by ANOVA (n = 3 per sample). (E) Flow cytometry analysis showed the apoptotic NSCs after 24 hours treatment with or without HSV-1 infection (0.2 MOI or 2 MOI) for 2 hours. (F) Bar graphs showed the percentage of late apoptotic cells. Data represent the mean +- SEM. *p < 0.05, **p < 0.01 by ANOVA (n = 3 per sample).

Supportive validation

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Experimental details: Fig 2 HSV-1-infected NSCs exhibited dysregulated neural differentiation. (A) Schematic diagram depicting the effect of HSV-1-infection on the NSCs in the term of neuronal differentiation. (B) Sample images of NSCs after 24 hour infection without or with HSV-1(0.2 MOI or 2 MOI) for 2 hours, immunostained for NSCs markers Nestin (green) and SOX2 (red) and DAPI (blue). The images were taken using an Olympus microscope. (C) Quantification of Nestin and SOX2. (D) Detection of the mRNA expression of SOX2 and Nestin (n = 3). (E) The expressions of MAP2 were identified by immunofluorescence staining, and the quantifications (F) for the percentage of MAP2+ were shown using Image J. Data represent the mean +- SEM. *p < 0.05, **p < 0.01 by ANOVA (n = 3 per sample). (G) Unsupervised hierarchical clustering of genes differentially expressed in NSCs on the basis of infection with or without HSV-1 (0.2MOI, 2MOI). (H) Gene Ontology (GO) biological process groups enriched in genes down-regulated in NSCs infected with the HSV-1(0.2 MOI), compared with the NSCs.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig 3 The neuroepithelial buds and cerebral organoids recapitulated early stages of human fetal brain development. (A) Schematic diagram depicting the effect of HSV-1-infection on the neuroepithelial buds and brain organoids in the areas of modelling the pathological features of developing human fetal brain, including injured neurogenesis, impaired neuronal differentiation, abnormal microglial activation, and dysregulated brain regionalization. (B) Schematic diagram of the neuroepithelial bud and cerebral organoid method and timing. Scale bars: 50mum. (C) The immunofluorescence staining for PAX6, TUJ, Nestin, and SOX2 in the neuroepithelial buds at the day 18. Scale bars: 25 mum. (D) Expressions of the specific brain regions markers (forebrain, PAX6; hindbrain ISL1) in brain organoids at the day 18; the cerebral organoids derived from hiPSCs at the day 45 showed the diverse neuron subtypes, including the mature neurons (MAP2), the astrocyte marker (GFAP), and microglia markers (Iba1). Scale bars: 25 mum. The images were taken Images were captured on a Leica TCS SP8 STED confocal microscope.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig 4 HSV-1 infection impaired neurogenesis in neuroepithelial buds. (A) The immunofluorescence staining for Nestin and SOX2 in the neuroepithelial buds at 18 days after 3 days with HSV-1 infection (Mock, Low infection, and High infection). Scale bars: 25 mum. The images were taken Images were captured on a Leica TCS SP8 STED confocal microscope. (B) Relative fluorescence intensity statistics of SOX2 and Nestin expressions were shown in different groups. Specifically, the expression of SOX2 was calculated by dividing the integrated optical density (IOD) with the total area of the nucleus, and the expression of Nestin was calculated by dividing the IOD with the total area of the cytoplasm. Images were prepared using ImageJ software (NIH, MD, USA). Data represent the mean +- SEM. *p < 0.05, **p < 0.01 by ANOVA (n = 3 per sample). (C) The mRNA expressions of Nestin and SOX2 were identified by RT-PCR. Data represent the mean +- SEM. *p < 0.05, **p < 0.01 by ANOVA (n = 4 per sample). (D) The immunofluorescence staining on the neuroepithelial buds was performed for detecting positive HSV-1 gE envelop protein at 18 day after 3 days with HSV-1 infection. Scale bars: 25 mum. (E) Bar graphs showing the percentages of HSV-1-positive cells in the neuroepithelial buds. Data represent the mean +- SEM from four experiments.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: FIGURE 4 PANX1 is differentially localized in the three embryonic germ layers. (A) Immunofluorescent evaluation of PANX1 (green) demonstrates primarily cell surface localization in iPSCs, ectoderm and mesoderm with lesser intracellular pools. Nuclei (Hoechst, blue); lineage markers (SOX2, Nestin, Brachyury, magenta); Actin (phalloidin, red). (B) In endoderm cells PANX1 is localized intracellularly where it partially overlaps with early endosomes (EEA1, red; SOX17, magenta). Inset: regions of interest zoomed in to highlight regional PANX1 localization. Brightness and contrast were equally adjusted across conditions in FIJI. Scale bar = 50 mum.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: FIGURE 6 Immunofluorescent analysis of PANX1-/- embryoid bodies. (A) Immunofluorescence of ectoderm (Nestin, yellow; PAX6, gray), mesoderm (Brachyury, green) and endoderm (SOX17, magenta) in control and PANX1-/- day 5 whole mount EBs. (B) After 14 days of differentiation, control and PANX1-/- EBs were cryosectioned and evaluated for germ lineage expression. Equal brightness contrast enhancements were made in FIJI for picture clarity. Nuclei (Hoechst, blue). Scale bar = 100 mum.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3 The presence of M1 macrophages and hyperglycemia changes the expression of CSC marker genes in PDEC. H6c7- pBp or H6c7- kras cells were cultivated in mono- or coculture with M1-polarized macrophages (MPhi) under normo- or hyperglycemic conditions (5 or 25 mM of d -glucose) for 2 or 5 days. The epithelial cells were separated from direct coculture with macrophages via CD11b-MACS depletion of M1-MPhi or harvested from monoculture and used for qRT-PCR analysis. The relative mRNA levels of Nanog ( A ) and Nestin ( B ) for both cell lines are depicted. They are normalized to the housekeeping gene GAPDH and presented as n -fold expression compared with the monocultured 5 mM sample from the equivalent cultivation timespan. Normally distributed data are presented as mean and standard error of mean. n = 4 for 2-day culture, n = 7 for 5-day culture. In ( B ), protein levels of Nestin are also depicted; Hsp90 was used as loading control. A representative of three independent experiments is shown.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2 Cx43 protein expression and localization in iPSCs and cells of each germ lineage. ( A ) Representative immunofluorescent confocal micrographs, demonstrating Cx43 (green) localization in control iPSCs (SOX2, iPSCs: purple) and after directed differentiation toward the three germ lineages (Nestin, ectoderm; Brachyury, mesoderm; SOX17, endoderm: purple) and nuclei (Hoechst, nuclei: blue). Cx43 forms large puncta (white arrows) at the cell surface indicative of gap junction plaques. Scale bars = 50 um. ( B ) Representative Western blots and ( C ) densitometric analysis of total Cx43 protein expression in PAX6-positive ectoderm, Brachyury-positive mesoderm, and SOX17-positive endoderm cells. * p < 0.05 compared to undifferentiated iPSCs. Data represent the standard error of the mean of 3-4 independent experiments.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Flow cytometric analysis for pluripotent and lineage markers expressed on day 6 of differentiation in the presence of drugs. Alexa 647-APC and Alexa 488 antibodies were used to double stain cells to look for pluripotency (Oct4+/Brachury-), mesendoderm (Oct4-/Bra+, Bra+/FoxA2-), endoderm (FoxA2+) and neurectoderm (NCAM+, NCAM+/nestin+). (A) Antibody controls using RA-treated cells. (B) Lineage-specific marker analysis.

14-9843-82

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