Mouse anti tuj1

Manufactured by BioLegend

Sourced in United States, United Kingdom

Mouse anti-Tuj1 is a monoclonal antibody that specifically recognizes the neuronal-specific class III beta-tubulin protein, also known as Tuj1. This antibody is a useful tool for identifying and characterizing neurons in both cell culture and tissue samples.

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Lab products found in correlation

Lsm 710 confocal microscope, by Zeiss (1 mentions) T8787 100ml, by Merck Group (1 mentions) Horseradish peroxidase conjugated goat anti mouse secondary antibody, by Merck Group (1 mentions) Normal mouse igg sc 2025, by Santa Cruz Biotechnology (1 mentions) Rabbit anti gfap, by Merck Group (1 mentions) Mouse anti ha, by Thermo Fisher Scientific (1 mentions) Horseradish peroxidase conjugated goat anti rabbit secondary antibody, by Thermo Fisher Scientific (1 mentions) Mouse anti flag, by Merck Group (1 mentions) Rabbit anti tet1, by Merck Group (1 mentions) Mouse anti tet1, by Active Motif (1 mentions) Trypsin, by Thermo Fisher Scientific (1 mentions) Facsaria iiu, by BD (1 mentions) Rabbit anti pax6, by BioLegend (1 mentions) Rnasin, by New England Biolabs (1 mentions) Bca assay kit, by Thermo Fisher Scientific (1 mentions) Mouse anti fus, by Santa Cruz Biotechnology (1 mentions) Hoechst, by Merck Group (1 mentions) Confocal laser scanning microscope, by Leica (1 mentions) Rabbit anti gfap, by Agilent Technologies (1 mentions) Vectashield hardset antifade mounting medium, by Vector Laboratories (1 mentions)

Close spelling variants of this product

Anti-Tuj1 (27 citations) Mouse anti-tubulin β3 (Tuj1, (4 citations) Mouse anti-Tuj1 antibody (3 citations) Anti-TUJ1 mouse IgG primary antibody (2 citations) Mouse monoclonal anti-β-III tubulin (Tuj1) (2 citations)

14 protocols using mouse anti tuj1

Multimodal Imaging of Neuron-Glia Interactions

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Transwells, containing neurons, astrocytes, and microglia, were fixed in 4% paraformaldehyde (PFA) (Electron Microscopy Science catalog no. 15710) in PBS for 30 min at RT followed by washing with PBS 1× and permeabilization with 0.3% Triton (Sigma-Aldrich, catalog no. T8787-100ML) in PBS. Cells were blocked with 3% BSA (Sigma-Aldrich, catalog no. A7030-100G) in PBS for 30 min at RT. Primary antibodies used were goat anti-Iba1 (Abcam, catalog no. AB5076; 1:500), mouse anti-Tuj1 (BioLegend, catalog no. 801201; 1:1000), and rabbit anti-S100B (Agilent, catalog no. Z031129-2; 1:1000). Primary antibodies were diluted in 3% BSA + 0.1% Triton in PBS and incubated at 4°C overnight. Next day, cells were washed 3× with PBS and stained with secondary antibodies Alexa 488, 568, and 647 (Life Technologies; 1:500). Secondary antibodies were added for 2 hours at RT followed by 2× washing with PBS. Transwell membranes with the stained cells were mounted onto glass slides (VWR catalog no. 488311-703) with mounting buffer (Electron Microscopy Science catalog no. 17984-25). Images were acquired with the Zeiss LSM 710 confocal microscope at a 20× magnification, curated with the Zeiss ZEN software, and processed with Imaris 9.2.0 (Bitplane, Zurich, Switzerland).

Trapecar M., Wogram E., Svoboda D., Communal C., Omer A., Lungjangwa T., Sphabmixay P., Velazquez J., Schneider K., Wright C.W., Mildrum S., Hendricks A., Levine S., Muffat J., Lee M.J., Lauffenburger D.A., Trumper D., Jaenisch R, & Griffith L.G. (2021). Human physiomimetic model integrating microphysiological systems of the gut, liver, and brain for studies of neurodegenerative diseases. Science Advances, 7(5), eabd1707.

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Indirect Immunofluorescence for Reovirus

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Primary antibodies used for indirect immunofluorescence to stain specific structures include: guinea pig anti-σNS for reovirus inclusions [112 (link)], T1L core-specific rabbit antiserum for reovirus cores [113 (link)], mouse anti-TUJ1 (BioLegend, 801201) for neurons, chicken anti-MAP2 (Abcam, ab92434) for dendrites, and mouse anti-non-phosphorylated neurofilament H (Millipore Sigma, SMI-32) for axons. Equal volumes of sera from rabbits immunized and boosted with T1L or T3D reovirus strains were mixed [114 (link)], adsorbed on rat CN culture to deplete non-specific antibodies, and used to stain reovirus-infected cells. Alexa Fluor conjugated secondary antibodies were used for visualization of antigens by indirect immunofluorescence. Anti-core serum was used at a 1:250 dilution and all other antibodies were used at a 1:1000 dilution.

Aravamudhan P., Raghunathan K., Konopka-Anstadt J., Pathak A., Sutherland D.M., Carter B.D, & Dermody T.S. (2020). Reovirus uses macropinocytosis-mediated entry and fast axonal transport to infect neurons. PLoS Pathogens, 16(2), e1008380.

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Antibody Characterization Protocol

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Rabbit anti-TET1 (Millipore, 09–872), mouse anti-TET1 (Active Motif, 91171), rat anti-TET1 (Active Motif, 61741, refer to as 5D6), mouse anti-HA (Invitrogen, 26183), mouse anti-Flag (sigma, F1804 and F7425), mouse anti-Tuj1 (Biolegend, 801201), rabbit anti-GFAP (Sigma, HPA056030) antibodies were purchased commercially. Rabbit anti-EGR1 antibody (sc-189), mouse anti-EGR1 antibody (sc-101033), rabbit normal IgG (sc-2027) and mouse normal IgG (sc-2025) were purchased from Santa Cruz Biotechnology. For western blot analysis, goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (Invitrogen, 65–6120) was used at a 1:5000 dilution, goat anti-mouse horseradish peroxidase-conjugated secondary antibody (Sigma, A8924) was used at a 1:10,000 dilution. The antibodies used for the IP and western blot experiments were summarized in Supplementary Data 6.

Sun Z., Xu X., He J., Murray A., Sun M.A., Wei X., Wang X., McCoig E., Xie E., Jiang X., Li L., Zhu J., Chen J., Morozov A., Pickrell A.M., Theus M.H, & Xie H. (2019). EGR1 recruits TET1 to shape the brain methylome during development and upon neuronal activity. Nature Communications, 10, 3892.

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Isolation and Purification of Cortical Cells

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E13.5 dorsal telencephalon was microdissected, avoiding the lateral ganglionic eminence and structures ventral to it. The cortex was separated from the meninges, and cortices from samples of the same genotype were pooled and sliced into small, uniformly sized pieces. Tissues were digested with 2.5% Trypsin (Invitrogen, Carlsbad, CA), then dissociated into single cells by repeated pipetting. Cells were fixed in 4% paraformaldehyde (PFA), incubated with primary antibodies, and then secondary antibodies. Each step was carried out in 4°C for 30 mins, with rotation. RNAsin (NEB, Ipswich, MA) was added to buffers to prevent RNA degradation (Hrvatin et al., 2014 (link)). Cells were sorted using FACS Aria IIU (BD).
Antibodies: Rabbit anti-PAX6 (Biolegend 901301, 1:1000), Mouse anti-TUJ1 (Biolegend 801202, 1:1000)

Chau K.F., Shannon M.L., Fame R.M., Fonseca E., Mullan H., Johnson M.B., Sendamarai A.K., Springel M.W., Laurent B, & Lehtinen M.K. (2018). Downregulation of ribosome biogenesis during early forebrain development. eLife, 7, e36998.

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Western Blotting of Cellular Proteins

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Cells were lysed with RIPA buffer (Westnet, BP-115-500) supplemented with fresh protease inhibitors (Roche, 11873580001). Lysates were incubated on ice for 15 min and cleared through centrifugation (16000 g) for 15 min at 4°C. Supernatants were collected and protein concentration were determined using BCA assay kit (Thermo Scientific Pierce, 23227). Western Blotting analysis were performed as described^{23 (link)}. After blocking, blots were incubated with primary antibodies overnight at 4°C. Primary antibodies were used as follows: mouse anti-FUS (1:200, Santa Cruz, SC-373698; validated in Supplemental Figure 7); rabbit anti-Nup62 (1:1000, Bethyl Laboratories, A304-942A; validated in Supplemental Figure 7); mouse anti-Tuj1 (1:1000, BioLegend, 801201); mouse anti-maltose binding protein (Cell Signalling Technology, 2396; validated in Supplemental Figure 7). Followed by washes, blots were incubated with secondary antibodies as described^{23 (link)} and visualized using Odyssey Infrared Imager (LiCor, 9120). Image J was used for densitometry measurements. Data collection and analysis were not performed blind to the conditions of the experiments.

Lin Y.C., Kumar M.S., Ramesh N., Anderson E.N., Nguyen A.T., Kim B., Cheung S., McDonough J.A., Skarnes W.C., Lopez-Gonzalez R., Landers J.E., Fawzi N.L., Mackenzie I.R., Lee E.B., Nickerson J.A., Grunwald D., Pandey U.B, & Bosco D.A. (2021). Interactions between ALS-linked FUS and nucleoporins are associated with defects in the nucleocytoplasmic transport pathway. Nature neuroscience, 24(8), 1077-1088.

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Immunocytochemistry of Neural Cells

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Cells were fixed in 4% PFA for 20 min. After permeabilization (ice cold methanol at −20°C for 20 min) and blocking (10% normal goat serum in PBS, for 2 h at room temperature) cells were incubated overnight at 4°C with primary antibodies: rabbit anti-GFAP (1:1000, DAKO), mouse anti-Tuj1 (1:500, Biolegend), mouse anti-STAT3 (1:200, cell signaling), rabbit anti-phospho-STAT3 (1:200, cell signaling), rabbit anti-p44/42 MAPK (1:200, cell signaling), rabbit anti-phospho-p44/42 MAPK (1,200, cell signaling). Cells were then washed and treated for 2 h with 1:500 dilution of Alexa-secondary antibodies. Finally, cell nuclei were stained with Hoechst (5 μg/μL, Sigma) for 20 min. Cells were mounted in vectashield hardset antifade mounting medium (Vector Labs). Images were obtained with either a high-resolution camera (Leica Microsystems LTd DFC340FX) using the microscope Leica DMRB or Leica confocal laser scanning microscope or IN Cell Analyzer 2000. At least three random fields per well were taken imaged the stained cells automatedly counted using IN Cell Analyzer software or manually counted using ImageJ (in cases where complex cellular morphology could not be accurately segmented using automated software).

Gialeli A., Spaull R., Plösch T., Uney J., Llana O.C, & Heep A. (2023). The miRNA transcriptome of cerebrospinal fluid in preterm infants reveals the signaling pathways that promote reactive gliosis following cerebral hemorrhage. Frontiers in Molecular Neuroscience, 16, 1211373.

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Immunocytochemical Analysis of Differentiated Neurons

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For immunocytochemical analysis, differentiated neurons were grown on a 16-well format chamber slide System Nunc™ Lab-Tek™ (Thermo Fisher Scientific), previously coated with poly-d-lysine (Sigma Aldrich) and laminin (Sigma Aldrich), and fixed in a 4% paraformaldehyde solution. Next, cells were permeabilized in 0.5% Triton X-100—PBS and blocked with a blocking solution (3% BSA in PBS) at RT. Immunostaining was performed by the addition of primary antibodies to co-stain the DA neuronal marker TH (rabbit anti-TH, Merck Millipore MAB318) and the class III member of the beta-tubulin family (mouse anti-TUJ1, Biolegend). Subsequently, cells were incubated with the appropriate fluorescently labeled secondary antibodies (goat anti-mouse Alexa Fluor 488-conjugated, goat anti-rabbit Alexa Fluor 555-conjugated, Thermo Fisher Scientific), nuclei were stained by adding NucBlue™ Fixed Cell Stain (Thermo Fisher Scientific), and the slides were mounted with Dako Fluorescence mounting medium. Images were acquired using a Leica SP8-X confocal microscope (Leica Microsystems, LAS-X acquisition software), with hybrid detectors and a 63X oil immersion objective.

Castelo Rueda M.P., Zanon A., Gilmozzi V., Lavdas A.A., Raftopoulou A., Delcambre S., Del Greco M F., Klein C., Grünewald A., Pramstaller P.P., Hicks A.A, & Pichler I. (2023). Molecular phenotypes of mitochondrial dysfunction in clinically non-manifesting heterozygous PRKN variant carriers. NPJ Parkinson's Disease, 9, 65.

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Immunofluorescence Staining Protocol

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The cells on the slide or tissue sections were blocked with 10% goat serum with 0.1% Tween-20 in PBS for 30 mins at room temperature (RT). Following this, sections were treated with primary antibodies in 3% goat serum with 0.1% Tween-20 in PBS at room temperature for 1 hr. The antibody we used were mouse anti-TUJ1 (BioLegend, 801202, 1:100). After washing with PBS, sections were incubated with secondary antibodies or Alexa Fluor^™ 568 Phalloidin (Thermo Fisher, A12380, 1:200) in 3% goat serum with 0.1% Tween-20 in PBS at RT for 1 hr. Finally, sections were washed and mounted with ProLong^™ Gold Antifade Mountant with DAPI (ThermoFisher, P36931). Staining was visualized on Leica DMi8 microscopy.

Rhim J.S., Webber M.M., Bello D., Lee M.S., Arnstein P., Chen L.S, & Jay G. (1994). Stepwise immortalization and transformation of adult human prostate epithelial cells by a combination of HPV-18 and v-Ki-ras.. Proceedings of the National Academy of Sciences of the United States of America, 91(25), 11874-11878.

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Immunocytochemical Analysis of Stem Cell Markers

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TCam-2 cells treated with or without ATRA were fixed in 4% paraformaldehyde. Fixed cells were permeabilized in 0.3% Triton X-100 in PBS containing 5% normal goat serum and subjected to immunocytochemical analyses as previously described (Kushwaha et al., 2015 (link)). Primary antibodies used for staining were goat anti-SOX2 (1:100) (catalog #sc17319; Santa Cruz Biotechnology), rabbit anti-NANOG (1:100) (#ab21624; Abcam), mouse anti-POU5F1 (1:200) (#sc5279; Santa Cruz), rabbit anti-BLIMP1 (#9115; Cell Signaling Technology), mouse anti-Nestin (#sc23927; Santa Cruz), rabbit anti-neurofilament (#ab-9034; Abcam), goat anti-TRKC (#ab188592; Abcam), and mouse anti-TUJ1 (#801201; BioLegend). Appropriate fluorescence-labeled secondary antibodies were used for visualization.

Kushwaha R., Jagadish N., Kustagi M., Mendiratta G., Seandel M., Soni R., Korkola J.E., Thodima V., Califano A., Bosl G.J, & Chaganti R.S. (2016). Mechanism and Role of SOX2 Repression in Seminoma: Relevance to Human Germline Specification. Stem Cell Reports, 6(5), 772-783.

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Immunocytochemistry: Protein Localization and Staining

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The slides were rehydrated in PBS and then blocked with 10% goat serum, 3% BSA, 0.4% Triton X-100 in PBS for one hour. The primary antibodies were incubated over-night diluted in the same solution at 4 °C. The following primary antibodies were used in this study: chicken anti-GFP 1:1000 (1020 AVES), rabbit anti-phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) antibody #9101 (Cell Signaling Technology, Danvers, MA, USA), rabbit anti-SOX2 1:500 (AB5603 Merck Millipore), mouse anti-Tuj1 1:500 (801202 Biolegend, California), rat anti-pH3 1: 250 (S28, Abcam ab10543), rabbit anti-caspase 3 1:500 (Asp175, CST 9661). The secondary antibodies used were as follows: anti-chicken, anti-rabbit, anti-mouse or anti-rat with fluorochromes (488, 568 or 647) at 1:500 (Alexa Fluor, Abcam, Cambridge). They were incubated for one hour in the blocking solution containing Hoechst (1:1000). F-actin staining was performed using phalloidin AlexaFluor568 (1:70) (Thermo Fisher, Waltham, MA, USA). The slides were washed, mounted (Thermo Scientific Shandon Immu-Mount) and imaged with a ZI Zeiss microscope equipped with an ApoTome or a confocal LSM 780.

Wilmerding A., Bouteille L., Rinaldi L., Caruso N., Graba Y, & Delfini M.C. (2021). HOXB8 Counteracts MAPK/ERK Oncogenic Signaling in a Chicken Embryo Model of Neoplasia. International Journal of Molecular Sciences, 22(16), 8911.

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