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Anti nestin

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Anti-Nestin is a laboratory reagent used to detect the presence of the nestin protein in cell samples. Nestin is an intermediate filament protein that is commonly expressed in stem cells and progenitor cells. Anti-Nestin can be used in various research applications, such as the identification and characterization of stem cells and progenitor cells.

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Lab products found in correlation

Anti sox1, by R&D Systems (2 mentions) Tryple express, by Thermo Fisher Scientific (1 mentions) Anti cleaved caspase 3, by Cell Signaling Technology (1 mentions) Anti grp78 bip, by Abcam (1 mentions) Anti neun, by Merck Group (1 mentions) Anti map2, by Merck Group (1 mentions) Anti sox2, by R&D Systems (1 mentions) Anti gadd153 chop, by Santa Cruz Biotechnology (1 mentions) Alexa fluor 488 555 594, by Thermo Fisher Scientific (1 mentions) Anti gfap, by Agilent Technologies (1 mentions) Hcs cellmask deep red stain, by Thermo Fisher Scientific (1 mentions) Anti tbr1, by Abcam (1 mentions) Hoechst 33342, by Dojindo Laboratories (1 mentions) Anti p62, by Abcam (1 mentions) Anti tuj1, by Fortrea (1 mentions) Alexa fluor 555 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions) Monoclonal rabbit anti th, by Merck Group (1 mentions) Anti arg 1, by BD (1 mentions) Anti gfp, by Abmart (1 mentions)

Spelling variants of this product

Nestin (22 citations) Mouse anti-Nestin (5 citations) Anti-Nestin (mouse clone 10C2, (2 citations) Rabbit anti-nestin (2 citations)

8 protocols using anti nestin

1

Neural Differentiation of Human iPSCs

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Human iPSCs were differentiated into haematopoietic cells as described previously17 (link). Neural cell differentiation was performed in accordance with previous protocols60 (link) with slight modifications. iPSC colonies were mechanically dissociated into small aggregate suspensions and then plated onto Costar six-well ultra-low-attachment plates (Corning, NY, USA) in DFK medium (hES medium with 10 μM SB431542, 2 μM Compound C and 1% penicillin/streptomycin) with 10 μM ROCK inhibitor (Y27632; Reagents Direct). Medium without ROCK inhibitor was changed on day 2, day 4, and day 6. On day 8, EBs were collected and plated on Matrigel-coated 12-well plates in DFN2D medium containing DMEM/F12, 50% Neurobasal medium, 0.5% N2 supplement, 0.5% B27 supplement, 1% L-alanyl-L-glutamine, 1% MEM nonessential amino acid solution, 1% glutamax-1,2-mercaptethanol, 1% penicillin/streptomycin and 20 ng/µl basic fibroblast growth factor. Medium was changed every 2 days for 6 days. On day 18, cells were dissociated using TryPLE Express (Life Technologies) and plated on 24-well plates. Passage was performed every 2 days. Immunohistochemistry was performed using the following primary antibodies: anti-Sox1 (R&D Systems) and anti-Nestin (eBiosciences).
Omori S., Tanabe H., Banno K., Tsuji A., Nawa N., Hirata K., Kawatani K., Kokubu C., Takeda J., Taniguchi H., Arahori H., Wada K., Kitabatake Y, & Ozono K. (2017). A Pair of Maternal Chromosomes Derived from Meiotic Nondisjunction in Trisomy 21 Affects Nuclear Architecture and Transcriptional Regulation. Scientific Reports, 7, 764.
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2

Immunocytochemistry for Stem Cell Characterization

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Immunocytochemistry was performed as described previously with some modifications18 (link). Cells were fixed with 4% paraformaldehyde/PBS and permeabilized with 0.5% Triton X-100/PBS for 15 min. Then, cells were blocked with 5% foetal bovine serum/PBS for 30 min. For primary antibodies, cells were incubated at 4 °C for 16 h with the respective antibodies, such as anti-SOX1 (1:100, R&D Systems), anti-SOX2 (1:100, R&D Systems), anti-PAX6 (1:100, Stemgent, San Diego, CA, USA), anti-Nestin (1:200, eBioscience, San Diego, CA, USA), anti-TUJ1 (1:1,500, Covance, Berkeley, CA, USA), anti-MAP2 (1:1,500, Millipore), anti-TBR1 (1:200, Abcam, Cambridge, UK), anti-GFAP (1:1,500, DakoCytomation, Carpinteria, CA, USA), anti-p62 (1:100, Abcam), anti-NeuN (1:100, Millipore), anti-cleaved caspase 3 (1:800, Cell Signaling Technology, Beverly, MA, USA), anti-GRP78/BIP (Abcam), or anti-GADD153/CHOP (Santa Cruz Biotechnology). Cells were washed with PBS and then incubated for 60 min with secondary antibodies such as Alexa Fluor 488, 555, 594, or 647 (Thermo Fisher Scientific). Nuclei were counterstained with Hoechst 33342 (Dojindo, Kumamoto, Japan). The cytoplasm was stained with HCS CellMask Deep Red Stain (Thermo Fisher Scientific).
Hirata K., Nambara T., Kawatani K., Nawa N., Yoshimatsu H., Kusakabe H., Banno K., Nishimura K., Ohtaka M., Nakanishi M., Taniguchi H., Arahori H., Wada K., Ozono K, & Kitabatake Y. (2020). 4-Phenylbutyrate ameliorates apoptotic neural cell death in Down syndrome by reducing protein aggregates. Scientific Reports, 10, 14047.
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3

Immunostaining Analysis of Cellular Markers

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In immunostaining analysis, non-specific binding was blocked with 3% bovine serum (Serotec, UK), Such binding underwent permeabilization for 30mins with 0.1% Triton X-100 in 1% BSA-PBS. The sections underwent the incubation overnight at 4°C with monoclonal rabbit anti-TH (1:500; Millipore), anti-GFP (1:500; Abmart), anti-iNOS (1:500; Proteintech), anti-Arg-1 (1:200; BD), anti-Nestin (1:100; eBioscience). Subsequently, they underwent the incubation with the relevant Alexa Fluor 488-conjugated secondary anti-body (1:1000; Invitrogen) or Alexa Fluor 555-conjugated secondary antibody (1:1000; Invitrogen) for 2hrs at ambient temperature. The nucleus was stained by DAPI (Invitrogen). The control section ran under the same protocol, but the major antibodies were omitted.
Tang Y., Han L., Bai X., Liang X., Zhao J., Huang F, & Wang J. (2020). Intranasal Delivery of Bone Marrow Stromal Cells Preconditioned with Fasudil to Treat a Mouse Model of Parkinson’s Disease. Neuropsychiatric Disease and Treatment, 16, 249-262.
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4

Immunohistochemical Analysis of Neural Markers

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After being deparaffinized and rehydrated, the immunohistochemistry of sections was performed as we described previously51 (link). Briefly, after treating with 0.3% Triton X-100 and 3% BSA and incubating at room temperature for 30 min, sections were incubated with the primary antibody anti-RAX (1:200; abcam, UK), anti-CHX10 (1:200; Santa Cruz; USA), anti-SOX2 (1:500; abcam), anti- β tubulin III (1:1000; Beyotime, Shanghai, China), anti-Ki67 (1:500; abcam) or anti-NESTIN (1:800; Thermo) in 1% BSA (overnight, 4 °C). The secondary antibodies cy3- or 488-conjugated (Jackson ImmunoResearch, West Grove, PA, USA) were then applied (1:200; 3 h). Before examination with a confocal laser scanning microscope (Leica, Germany), the sections were counterstained with DAPI (Sigma-Aldrich, St. Louis, MO, USA).
Gao L., Chen X., Zeng Y., Li Q., Zou T., Chen S., Wu Q., Fu C., Xu H, & Yin Z.Q. (2016). Intermittent high oxygen influences the formation of neural retinal tissue from human embryonic stem cells. Scientific Reports, 6, 29944.
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5

Immunostaining of Neural Progenitor Cells

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Neurospheres were allowed to sediment on poly-D-lysine/laminin-coated coverslips. Cells were fixed with ice-cold paraformaldehyde (4%, w/v) for 20 min, then permeabilized with 0.25% Triton X-100 for 5 min, blocked with 5% normal goat serum, and incubated overnight at 4 °C with anti-nestin (1:500, Thermo Scientific, PA5-17428) and anti-doublecortin (1:1000, Thermo Scientific, 2Q178) antibodies. Then, cells were incubated with Alexa Fluor 488-conjugated anti-rabbit (1:500, Life technologies, A11008) and Alexa Fluor 546-conjugated anti-mouse (1:500, Life technologies, A11003) antibodies at RT for 1 h, and the nuclei were counterstained for 5 min with 4′,6-diamidino-2-phenylindole (DAPI). Immunofluorescence was visualized using a Zeiss LSM 800 II Microscope.
Campero-Romero A.N., Real F.H., Santana-Martínez R.A., Molina-Villa T., Aranda C., Ríos-Castro E, & Tovar-y-Romo L.B. (2023). Extracellular vesicles from neural progenitor cells promote functional recovery after stroke in mice with pharmacological inhibition of neurogenesis. Cell Death Discovery, 9, 272.
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6

Xenograft Tumor Transplantation and Characterization

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Tumor transplantation experiments and procedures were approved by the University of Utah Institutional Animal Care and Use Committee and were performed as described [18 (link), 28 (link)]. Non-obese diabetic/severe-combined immunodeficient IL-2Rg-null mice of both sexes were used at the age of 6–10 weeks. Transplantations required 2 × 106 BT142 cells for subcutaneous injections and 2 × 104 cells for intracranial injections. Bioluminescent imaging with inhalant isoflurane was performed essentially as described [18 (link), 28 (link)]. Bioluminescent intensity as a surrogate of tumor volume was quantitatively analyzed using LivingImage software (Xenogen, Alameda, CA).
Tumors were harvested 6–8 weeks after injection for formalin fixation and paraffin embedding. Histological assessment and immunohistochemistry analysis were as previously described [18 (link)]. Primary antibodies were diluted as follows: 1:10,000 anti-5hmC (Invitrogen), 1:25 anti-Nestin (Invitrogen), and 1:500 anti-GLUD (Invitrogen). To confirm IDH1R132H heterozygosity in the resultant tumor, genomic DNA was extracted from paraffin-embedded intracranial tumors using DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany). PCR amplification and DNA sequencing were performed to distinguish YFP* from YFP‑IDH1 transgene [17 (link)].
Tiburcio P.D., Gillespie D.L., Jensen R.L, & Huang L.E. (2020). Extracellular glutamate and IDH1R132H inhibitor promote glioma growth by boosting redox potential. Journal of neuro-oncology, 146(3), 427-437.
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7

Comprehensive Immunohistochemical Analysis of Neural Markers

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Samples were collected and fixed in 10% formalin for 1 h at room temperature followed by embedding in paraffin. Then hemotoxylin/eosin (H&E) staining and immunofluorescence were performed. After blocking, slides were treated with primary antibodies in 5% bovine serum albumin with concentration as follows: anti-N-cadherin (rabbit, Cell Signaling 13116, 1:300), anti-vimentin (rabbit, Cell Signaling 5741, 1:300), anti-Ki-67 (mouse, cell signaling, 1:200), anti-cleaved Caspase 3 (rabbit, Invitrogen, 1:200), anti-Sox2 (mouse, Invitrogen, 1:200), anti-Oct4 (mouse, Invitrogen, 1:200), anti-FoxG1 (rabbit, Abcam, 1:200), anti-TUJ1 (rabbit, Abcam, 1:200), anti-Auts2 (rabbit IgG, Proteintech, 1:50), anti-Islet1 (rabbit IgG, Proteintech, 1:50), anti-Nell2 (rabbit IgG, Proteintech, 1:50), anti-GFAP (rabbit IgG, Abcam, 1:200), anti-H3-di-K4 (rabbit, Abcam, 1:200), anti-LSD1 (rabbit, Life technology, 1:300), anti-CD133 (rabbit, Cell Signaling, 1:300), anti-EGFR (rabbit, cell signaling, 1:300), anti-Tubulin (rabbit, Invitrogen, 1:200), anti-actin (rabbit, Invitrogen, 1:200), anti-nestin (mouse, Invitrogen, 1:200). Secondary antibodies include Alexa Fluor 488 rabbit, Alexa 488 Fluor mouse, Alexa 594 Fluor rabbit, and Alexa 594 Fluor mouse IgG (Invitrogen, 1:500). Tranylcypromine was from Selleck Chemicals. The images of immunostaining were acquired by an Olympus fluorescent microscope.
Huang J., Liu F., Tang H., Wu H., Li L., Wu R., Zhao J., Wu Y., Liu Z, & Chen J. (2017). Tranylcypromine Causes Neurotoxicity and Represses BHC110/LSD1 in Human-Induced Pluripotent Stem Cell-Derived Cerebral Organoids Model. Frontiers in Neurology, 8, 626.
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8

Antibody Generation and Characterization

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All reagents were of chemical grade and purchased from Sigma-Aldrich (MO, USA) unless specifically indicated. Antibodies against α4 (181-192), α7 (179-190), β2 (190-200) andα7 (1-208) nAChR fragments and rabbit cyt c-specific antibodies were generated using methods previously developed in the authors . The antibodies were biotinylated according to standard procedures [Citation35] . The antibodies anti-Nestin, anti-mouse Fab Alexa 555-conjugated and anti-rabbit Fab Alexa 488-conjugated as well as the kits for IL-1β, IL-6, IL-10 and TNF-α determination (Invitrogen, MA, USA) and NeutrAvidin-peroxidase conjugate (Molecular Probes, OR, USA) were purchased from ALT Ukraine, an official representative of Thermo Fisher Scientific in Ukraine. In addition, BrdU (category number: 5002) and anti-BrdU (category number: B8434) were purchased from Sigma-Aldrich, and antibodies against Aβ 1-42 (category number: ab2539) were purchased from Abcam (Cambridge, UK).
Skok M., Deryabina O., Lykhmus O., Kalashnyk O., Uspenska K., Shuvalova N., Pokholenko I., Lushnikova I., Smozhanyk K., Skibo G, & Kordyum V. (2022). Mesenchymal stem cell application for treatment of neuroinflammation-induced cognitive impairment in mice. Regenerative medicine, 17(8).
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