TPX2 antibody was a kind gift from Oliver Gruss24 (link). Anti-RanGTP antibody (AR-12) was a kind gift from Ian Macara37 (link). Mouse anti-α-tubulin monoclonal antibody DM1A (T6199) and Duolink reagents were from Sigma (St Louis, MO). Mouse anti-β-III-tubulin antibody TUJ1 (MMS-435P) and mouse anti-neurofilament monoclonal antibody SMI312 (SMI-312R) were from Covance (Princeton, NJ). Mouse anti-γ-tubulin antibody GTU-88 (GTX11316), HRP-labeled goat-anti-rabbit IgG antibody (GTX213110), and HRP-labeled goat-anti-mouse IgG antibody (GTX213111) were from GeneTex (Irvine, CA). Mouse anti-actin antibody C4 (MAB1501), mouse anti-GAPDH antibody 6C5 (MAB374), and rabbit anti-MAP2 polyclonal antibody (AB5622) were from Millipore (Billerica, MA). Rabbit anti-Ran polyclonal antibody (ab31118) and rabbit anti-importin β1 (ab45938) were from Abcam (Cambridge, UK). Mouse anti-importin-α antibody (sc-55538) was from Santa Cruz Biotechnology (Santa Cruz, CA). Dylight-conjugated secondary antibodies were from Jackson ImmunoResearch (West Grove, PA). Alexa Fluor-conjugated secondary antibodies were from Life Technologies (Carlsbad, CA).
Tuj1 mms 435p
Manufactured by Fortrea
The TUJ1 (MMS-435P) is a laboratory equipment product designed for a specific function. It is a compact and portable device intended for use in controlled laboratory settings. The core function of the TUJ1 (MMS-435P) is to perform a specialized task, but the details of its intended use are not available in this concise, unbiased, and factual description.
Automatically generated - may contain errors
Lab products found in correlation
B27 supplement, by Thermo Fisher Scientific (2 mentions)
N2 supplement, by Thermo Fisher Scientific (2 mentions)
Alexa fluor tagged secondary antibodies, by Thermo Fisher Scientific (2 mentions)
Prism 5, by GraphPad (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Neurobasal medium, by Thermo Fisher Scientific (2 mentions)
Low glucose dmem, by Thermo Fisher Scientific (2 mentions)
Nestin ab6142, by Abcam (2 mentions)
Gfap g9269, by Merck Group (2 mentions)
O4 mab345, by Merck Group (2 mentions)
Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Dylight conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Ab5622, by Merck Group (1 mentions)
Ab45938, by Abcam (1 mentions)
Mouse anti gapdh antibody 6c5 mab374, by Merck Group (1 mentions)
Smi312 smi 312r, by Fortrea (1 mentions)
Virapower mix, by Thermo Fisher Scientific (1 mentions)
Actin a3853, by Merck Group (1 mentions)
Plenti4 to v5 dest, by Thermo Fisher Scientific (1 mentions)
11 protocols using tuj1 mms 435p
1
Immunofluorescence Microscopy Antibodies
2
Stem Cell Characterization Protocol
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The following antibodies were purchased: GFAP (Z0334) from DAKO; Nestin (N1602) from IBL; TuJ1 (MMS-435P) from Covance; Sox2 (MAB2018) from R&D Systems; KLF4 (4038), ERK (4695), and p-ERK (9106) from Cell signaling; Actin (A3853) from Sigma-Aldrich; p53 (sc-126), c-Myc (sc-40) from Santa Cruz. The following plasmids were purchased: pLenti4/TO/V5-DEST and ViraPower mix from Invitrogen; pLVX-Tet-On 3G (631354); pLM-mCherry-KLF4 (23243) pCMV-VSV-G (8454) and pCMV-dR8.2 dvpr (8455) from Addgene; KLF4 ShRNA (RHS4533-NM_004235) and EGR1 ShRNA (RHS4533-NM_001964) from Openbiosystem.
3
Immunocytochemistry and Immunohistochemistry Protocols
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Cells fixed in 4% paraformaldehyde (PFA) for 30 min at room temperature were used for immunocytochemistry (ICC). PFA-fixed tissue was embedded in optimal cutting temperature compound (OCT) and cryostat cut into tissue sections (12 µm) for IHC. The fixed cells or tissue sections were blocked in 5% donkey serum plus 0.1% Triton X-100 in PBS (PBS-T) for 1–2 h at room temperature, and then incubated overnight at 4 °C with the following primary antibodies: S100β (S2532, Sigma, 1:200), MAG (34–6200, Invitrogen, 1:100), Stat1 (14994, Cell Signaling, 1:200), beta-III tubulin (TuJ1, MMS-435P, Covance, 1:250), P0 (NB100-1607, Novus, 1:50). After 3 washes with PBS, the cells or tissue sections were incubated with corresponding fluorescence-conjugated secondary antibodies (1:1,000; Jackson Immunoresearch, PA, USA) for 1 h at room temperature. The images were captured using a fluorescence microscope (Leica).
4
Immunofluorescence Staining of Tissue Sections
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Cryostat sections were incubated with blocking buffer (2.5% BSA, 0.01% Triton-X100) for 1 h at RT and incubated with primary antibody diluted in blocking buffer overnight at 4°C. Sections were washed with PBS and incubated with secondary antibody for 2 h at RT, washed with PBS and mounted using Fluoro-Gel (Electron Microscopy Services #17985-11). Antibodies used were: Abcam: CTIP2 (ab18465) 1:300; BD Transduction Labs: N-cadherin (610920) 1:500; Cell Signaling Technology: Cleaved caspase-3 (#9661) 1:200, Connexin-43 (#3512) 1:300, Ki67 (#9129) 1:400, phosH3 (#9706S) 1:300; Chemicon: Sox2 (AB5603) 1:400, MAP2 (MAB3418) 1:400, TBR2 (AB9618) 1:400; Covance: Tuj1 (MMS-435P) 1:400, Pax6 (PRB-278P) 1:400. The Pax6 mAb (1:200) developed by A. Kawakami was obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, IA 52242. Sections were incubated in the relevant secondary antibody conjugated to Alexa-Fluor 488 nm, 568 nm or 647 nm (Molecular Probes/Invitrogen) and counter-stained with DAPI (4’,6-diamidino-2-phenylindole) and/or SYTOX® green nucleic acid stain (Molecular Probes/Invitrogen), prior to mounting with Fluoro-Gel.
5
Immunocytochemical Characterization of Neural Progenitor Cells
6
Immunofluorescence Staining of Neural Cells
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For staining, cell cultures were fixed in 4% paraformaldehyde. Species-specific Alexa dye conjugate secondary antibodies were used (molecular probes). Antibodies for immunofluorescence staining were the following: FOXA2 (SC-6554) from Santa Cruz Biotechnology; Lmx1a (AB10533) from Millipore; MAP2 (M1406) from Sigma-Aldrich; NURR-1 (PP-N1404–00) from Perseus Proteomics; tyrosine hydroxylase (P40101–150) from Pel-Freez; TUJ1 (MMS-435P) from Covance.
7
Immunofluorescence Staining of Neural Progenitors
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Neural progenitor cells grown on poly-L-ornithine/laminin coated 4-well chamber plates were fixed in 4% paraformaldehyde for 15 min at room temperature, permeabilized with 0.3% Triton-X-100 in Triton Buffer Solution (TBS) for 10 min, blocked with 2% FBS in TBS-0.05% Tween-20 for 20 min and stained with primary antibodies in blocking buffer at 4°C overnight. Primary antibodies included: anti-Nestin (611658; BD Pharmingen); anti-SOX2 (561469; BD Pharmingen); anti-RFP (600-401-379; Rockland); anti-ki67-Alexa fluor-488 (51-9007231; BD Stemflow Human neural Lineage analysis Kit); TuJ1 (MMS-435P, Covance); GFAP (ab7779, Abcam). The next day, the cells were stained with secondary antibodies (Alexa fluor 568 donkey anti-rabbit; Alexa fluor 488 donkey anti-mouse; Life Technologies, Inc.) in blocking buffer for 45 min and nuclei were counterstained with DAPI. Images were acquired using an Olympus FV1000 confocal microscope. Live culture images were also acquired using an Inverted Axioscope and AxioCam MRm (Carl Zeiss, Inc.). Image assembly was performed using Adobe Photoshop CS5 (Adobe Systems, Inc.).
8
Immunocytochemistry Protocol for Neuronal Markers
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Cultures were fixed with 4% paraformaldehyde. Cells were washed 3 times with phosphate-buffered saline (PBS) after each step. The cells were treated with -20°C ethanol/acetic acid solution, permeabilized with a 0.2% Triton-X 100 solution, and blocked with 1% cold fish gelatin (Sigma, St. Louis, MO). The cells were incubated overnight at 4°C with primary antibodies for Oct3/4 (sc-5279, 1:200; Santa Cruz Biotechnology, Santa Cruz, CA), NeuN (MAB377 1:200; Millipore), β3 Tubulin (Tuj-1, MMS-435P, 1:200; Covance, Princeton, NJ), neurofilament (AB9568, 1:100; Millipore), synaptosomal-associated protein 25 (SNAP-25, AB5871P, 1:100; Millipore), synapsin 1 (51-5200, 1:200; Life Technologies, Grand Island, NY), FOXG1 (1:200, Abcam, Cambridge, MA), and GFP (1:100, Polysciences, Inc., Warrington, PA). The cells were washed with PBS after the overnight antibody incubation. The corresponding secondary antibodies were applied at 1:100 for 1 hr at room temperature (Jackson ImmunoResearch, West Grove, PA), Cells were washed with PBS and Hoechst 33342 was applied (1:20,000) and washed off. The cells were cover-slipped with Vectashield mounting media (Vector Laboratories, Burlingame, CA).
9
Immunocytochemical Characterization of Neural Progenitor Cells
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Immunocytochemistry was performed on trypsinized NPCs grown in 50 µg/mL poly-D-lysine-coated and 10 µg/mL fibronectin-coated 24-well plates containing defined medium [(5:3 mixture of DMEM low glucose (Life Technologies): neurobasal medium (Life Technologies), 0.5 mM 2-mercaptoethanol, 2mM L-glutamine, 5 IU of penicillin, and 5 µg/ml streptomycin (life technologies) supplemented with 1% N2 supplement (Life Technologies) and 2% B27 supplement (Life Technologies) without growth factors. After 5 days in culture, cells were fixed and stained with primary antibodies (Nestin: ab6142 (Abcam), GFAP: G9269 (Sigma-Aldrich), Tuj1: MMS-435P (Covance), and O4: MAB345 (Millipore)) at 4°C overnight. For fluorescence detection, Alexa Fluor-tagged secondary antibodies (Molecular Probes) were used and cells were counterstained with DAPI. Images were acquired using Metamorph software on a Nikon Eclipse TE300 microscope equipped with an optical camera (Optronics). Investigators were blinded to mouse genotypes and percentages of positive cells were calculated using the total number of cells in each image (DAPI nuclear stain). Data were analyzed with Prism 5 software (GraphPad Software) using a two-way ANOVA test as all data met the common assumptions (normally distributed, equal variances, independent samples) required for this test.
10
Immunocytochemical Analysis of Stem Cell Markers
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ALP staining was performed using the Alkaline Phosphatase Detection Kit (Chemicon) as instructed by the manufacturer. Immunocytochemistry was performed using standard protocol. Briefly, cells were fixed in 2% paraformaldehyde (Sigma-Aldrich), washed three times with PBS, and then incubated in PBS containing 0.1% Triton X-100 and 3% skim milk in PBS for 1 h at room temperature. The cells were then incubated with the following primary antibodies at 4°C overnight: Nanog (ab21603, 1:500, Abcam); Oct4 (sc-5279, 1:100, Santa Cruz); Sox2 (AB5603, 1:500, Millipore); SSEA1 (sc-21702, 1:100, Santa Cruz); Tuj-1 (MMS-435P, 1:500, Covance); CD31 (sc-1506-R, 1:100, Santa Cruz); and IgG (sc-2025, 1:500, Santa Cruz). After washing three times with PBS, cells were incubated with secondary antibodies: Alexa Fluor 488 goat anti-mouse IgG (1:1000, Molecular Probes) and Alexa Fluor 488 goat anti-rabbit IgG (1:1000, Molecular Probes) for 2 h at room temperature. Nuclei were detected by propidium iodide (PI) (Vector Laboratories, Inc.) staining. Images were analysed by confocal microscopy (FV1000; Olympus).
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