Mouse anti pax6

Manufactured by Developmental Studies Hybridoma Bank

Sourced in China, United States

Mouse anti-PAX6 is a monoclonal antibody developed by the Developmental Studies Hybridoma Bank. It recognizes the PAX6 protein, which is a transcription factor involved in the development of the eye, pancreas, and nervous system. The antibody can be used for detection and analysis of the PAX6 protein in various research applications.

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Lab products found in correlation

Goat anti gfp, by Rockland Immunochemicals (2 mentions) Lsm 780, by Zeiss (2 mentions) Ab5603, by Merck Group (2 mentions) Ab10543, by Abcam (2 mentions) Alexa fluor 568 phalloidin, by Thermo Fisher Scientific (2 mentions) Shandon immu mount, by Zeiss (2 mentions) Lsm 510 meta confocal microscope, by Zeiss (2 mentions) Rabbit anti gfp, by Thermo Fisher Scientific (2 mentions) Goat anti mouse igg cy3, by Jackson ImmunoResearch (1 mentions) Donkey anti goat igg alexa488, by Thermo Fisher Scientific (1 mentions) Odyssey clx imaging system, by LI COR (1 mentions) Prism 7, by GraphPad (1 mentions) Mouse anti β actin, by Merck Group (1 mentions) Rabbit anti gfap, by Merck Group (1 mentions) Mouse anti tuj1, by Merck Group (1 mentions) Mouse anti o4, by Merck Group (1 mentions) Goat anti sox2, by Santa Cruz Biotechnology (1 mentions) Mouse anti nestin, by Developmental Studies Hybridoma Bank (1 mentions) Rabbit anti sox2, by Merck Group (1 mentions) Mouse anti γ tubulin, by Merck Group (1 mentions)

Close spelling variants of this product

Anti-Pax6 (3 citations) Mouse monoclonal anti-Pax6 (2 citations)

11 protocols using mouse anti pax6

Cryostat Immunofluorescence Staining Protocol

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Cryostat sections were rinsed in PBS at 37°C for 3 min followed by 3 min in cold water. Subsequently the sections were incubated in 20 mM lysine in 0.1 M sodium phosphate (pH 7.4) for 30 min at room temperature before being rinsed in PBS three times for 10 min. The tissue was permeabilized with 0.1% Triton in PBS for 30 min at room temperature and then washed again three times with PBS for 10 min. To prevent unspecific binding of the antibody the tissue was blocked with 10% fetal calf serum (FCS) in PBS for 1 h. Goat anti-GFP (1:400; Rockland), rabbit anti-Calbindin D-28K (CB38a; Swant), mouse anti-Pax6 (2 μg/ml; Developmental Studies Hybridoma Bank) were dissolved in 10% FCS/PBS and incubated overnight at 4°C. After three washes in PBS, 10% FCS in PBS was applied again for 1 h, followed by the incubation with donkey anti-rabbit IgG-Cy3 (1:250; Molecular Probes), donkey anti-goat IgG-Alexa488 (1:250; Molecular Probes/Invitrogen) and goat anti-mouse IgG-Cy3 (1:250; Jackson ImmunoResearch) dissolved in 10% FCS in PBS for 90 min at room temperature. The tissue was rinsed 5 times in PBS for 12 min and then mounted in Celvol (Celanese). The staining of cryostat sections was analyzed with an upright microscope equipped with fluorescence optics (Olympus BX51).

Baeriswyl T., Schaettin M., Leoni S., Dumoulin A, & Stoeckli E.T. (2022). Endoglycan Regulates Purkinje Cell Migration by Balancing Cell-Cell Adhesion. Frontiers in Neuroscience, 16, 894962.

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Immunofluorescence Staining of Tissue Sections

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Slides were rehydrated in PBS then blocked with 10% goat serum, 3% BSA, 0,4% Triton X-100 in PBS for one hour. Primary antibodies were incubated overnight 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) 1:250 (Cell signaling #9101), rabbit anti-SOX2 1:500 (AB5603 Merck Millipore), mouse anti-TUJ1 1:500 (801202 Biolegend), rat anti-pHH3 1: 250 (S28, Abcam ab10543), rabbit anti-cleaved-caspase 3 1:500 (Asp175, CST 9661), mouse anti-PAX6 1:500 (Developmental Studies Hybridoma Bank), Phalloidin AlexaFluor 568 1:40 (ThermoFisher). The secondary antibodies used were: anti-chicken, anti-rabbit, anti-mouse or anti-rat with conjugated fluorochromes (Alexa Fluor 488, 568 or 647; ThermoFisher) at 1:500. Sections were incubated for one hour in the blocking solution containing Hoechst dye (1:1000). Slides were washed, mounted (Thermo Scientific Shandon Immu-Mount) and imaged with a Zeiss microscope Z1 Apotome or a confocal LSM 780.

Wilmerding A., Bouteille L., Caruso N., Bidaut G., Etchevers H.C., Graba Y, & Delfini M.C. (2021). Sustained experimental activation of FGF8/ERK in the developing chicken spinal cord models early events in ERK-mediated tumorigenesis. Neoplasia (New York, N.Y.), 24(2), 120-132.

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Western Blot Analysis of PAX6 and β-Actin

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For western blots, cellular lysate was subjected to 10% SDS-PAGE and immunoblotted with mouse anti-PAX6 1:100 (Developmental Studies Hybridoma Bank) and mouse anti-beta-ACTIN 1:4,000 (Sigma-Aldrich). The secondary antibody was either anti-mouse immunoglobulin G (IgG) horseradish peroxidase (HRP) conjugate (R&D Systems) at a working dilution of 1:4,000 to 1: 10,000, or anti-mouse IgG (H&L) (goat) Antibody DyLight 680 Conjugated (Rockland) at a dilution of 1:10,000. Blots were detected using Li-Cor Odyssey CLX imaging system, and analysis was undertaken using Image Studio software. For western blots of islets, 100 islets collected from at least three different animals of the same genotype were used for each well. Data and statistical analysis was done using Graph Pad Prism 7.0.

Yongblah K., Alford S.C., Ryan B.C., Chow R.L, & Howard P.L. (2018). Protecting Pax6 3′ UTR from MicroRNA-7 Partially Restores PAX6 in Islets from an Aniridia Mouse Model. Molecular Therapy. Nucleic Acids, 13, 144-153.

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Immunocytochemistry of Neural Cell Markers

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The following antibodies were used: mouse anti-Tuj1 (1:50, Millipore), mouse anti-microtubule-associated protein (MAP) 2 (1:200; Sigma–Aldrich), rabbit anti-GFAP (1:200; Millipore), mouse anti-O4 (1:100, Millipore), mouse anti-CNPase (1:100, Millipore), goat anti-Sox2 (1:100, Santa Cruz), mouse anti-Nestin (1:100; Developmental Studies Hybridoma Bank, Beijing, China), mouse anti-Pax6 (1:100; Developmental Studies Hybridoma Bank), donkey Alexa-555 anti-mouse (1:1000; Invitrogen), and donkey Alexa-555 anti-rabbit (1:1000; Invitrogen). Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI). The slide-mounted, stained samples were observed using an LSM 510 META confocal microscope (Zeiss, Oberkochen, Germany) with excitation wavelengths of 543, 488, and 405 nm. The channel signals were collected sequentially. Collected images were assembled using Adobe Photoshop (Adobe Systems, San Jose, CA).

Yao H., Gao M., Ma J., Zhang M., Li S., Wu B., Nie X., Jiao J., Zhao H., Wang S., Yang Y., Zhang Y., Sun Y., Wicha M.S., Chang A.E., Gao S., Li Q, & Xu R. (2015). Transdifferentiation-Induced Neural Stem Cells Promote Recovery of Middle Cerebral Artery Stroke Rats. PLoS ONE, 10(9), e0137211.

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Immunohistochemical Staining of Mouse Tissue

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Immunohistochemistry of coronal cryosections was performed as previously described (Burnett et al., 2017 (link)). Primary antibodies were used at the following concentrations: rabbit anti-PAX2 (1:450, Covance Research Products), mouse anti-PAX6 (1:50, Developmental Studies Hybridoma Bank), sheep anti-CHX10 (1:600, Exalpha Biologicals), mouse anti-MITF (1:1500, Abcam), rabbit anti-OTX2 (1:600, Upstate Biotechnology), mouse anti-COUPTFII (1:300, R & D Systems), goat anti-SOX1 (1:100, R & D Systems), rabbit anti-SOX2 (1:300, EMD Millipore), mouse anti-γ-TUBULIN (1:1000, Sigma-Aldrich), rabbit anti-ARL13B (1:3000, T. Caspary, Emory University). Images were taken with either a Zeiss Axioplan 2 or a Keyence BZ-×710 fluorescence microscope. High-magnification images of cilia are maximum intensity projections from z-stacks taken on a Zeiss LSM510 confocal microscope.

Lupu F.I., Burnett J.B, & Eggenschwile J.T. (2017). Cell cycle-related kinase regulates mammalian eye development through positive and negative regulation of the Hedgehog pathway. Developmental biology, 434(1), 24-35.

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Fluorescent Immunohistochemistry of Tissue Sections

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Fluorescent immunohistochemistry was performed on cryosectioned tissue as previously described51 (link). Antibodies utilized for fluorescent immunohistochemistry are as follows: goat anti-GFP (1:500; Rockland Immunochemicals, Limerick, PA), rabbit anti-GFP (1:1000; Invitrogen, Waltham, MA), mouse anti-Glutamine synthase (GLUL) (1:200; BD Biosciences, San Jose, CA), mouse anti-KI67 (1:200; BD Biosciences, San Jose, CA), rabbit anti-LHX2 (1:1500; generated in house with Covance, Princeton, NJ), mouse anti-NFIA (1:200; CDI Laboratories), mouse anti-P27^Kip1 (1:200; Invitrogen), mouse anti-PAX6 (1:200; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA), rabbit anti-phosphohistone H3 (PHH3) (1:200; Millipore, Billerica, MA). Secondary antibodies used were as follows: AlexaFluor488 conjugated donkey anti-goat IgG (1:500; Jackson Immunoresearch, West Grove, PA), AlexaFluor488 conjugated donkey anti-rabbit IgG (1:500; Jackson Immunoresearch, West Grove, PA), AlexaFluor594 conjugated donkey anti-rabbit IgG (1:500; Jackson Immunoresearch, West Grove, PA), AlexaFluor594 conjugated donkey anti-mouse IgG (1:500; Jackson Immunoresearch, West Grove, PA). All section immunohistochemical data shown was imaged and photographed on a Zeiss Meta 510 LSM confocal microscope.

de Melo J., Clark B.S, & Blackshaw S. (2016). Multiple intrinsic factors act in concert with Lhx2 to direct retinal gliogenesis. Scientific Reports, 6, 32757.

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Immunofluorescence Analysis of Neural Markers

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Cryosections (10 μm thick) were subject to antigen retrieval by microwaving for 20 min in 10 mM sodium citrate buffer. After blocking with 20% donkey serum, sections were incubated with primary antibodies overnight at 4 °C. The primary antibodies used were mouse anti-Pax6 (Developmental Studies Hybridoma Bank 1:100), rabbit anti-Cdca7 (Proteintech 1:100), goat anti-GFP and rat anti-BrdU (5-Bromo-2′-deoxyuridine, 1:200 Abcam), rabbit anti-Tbr1 and rabbit anti-Tbr2 (Abcam 1:100), 4′,6-Diamidino-2-Phenylindole (DAPI, Invitrogen 1:1000). Secondary antibodies were conjugated with Alexa Fluor 488 or 568 (Invitrogen). Fluorescent and confocal images were acquired with a Leica DM5500B microscope and a Nikon A1R confocal microscope, respectively.

Huang Y.T., Mason J.O, & Price D.J. (2017). Lateral cortical Cdca7 expression levels are regulated by Pax6 and influence the production of intermediate progenitors. BMC Neuroscience, 18, 47.

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Immunohistochemical Analysis of Embryonic Mouse Tissues

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For immunohistochemistry, heterozygous mice were fixed with 4% paraformaldehyde in PBS by intracardial perfusion. Tissues were post-fixed for 1h to overnight at 4°C. E14.5 mice were dated with E0.5 at time of plug detection. Embryos were drop fixed in 4% paraformaldehyde immediately after dissection. After post-fix, all tissue was washed extensively with PBS, cryopreserved in 30% sucrose in PBS overnight, embedded in OTC (Sakura Finetek, Torrance, CA), and frozen. Sections were cut at 20 μm on a cryostat and placed on slides. For immunostaining, sections were blocked in 10% goat serum and 0.25% triton-X in PBS for 1 h at RT. Sections were incubated with primary antibodies in block overnight at 4°C. Slides were washed 4 × 5 min in PBS containing 0.1% triton-X. Detection of antibodies was carried out using Alexa-fluor secondary antibodies (Life Technologies) diluted 1: 500 in blocking solution. Sections were washed, as above, and coverslipped. The following primary antibodies used were: rabbit anti-GFP (1:1,000, Life Technologies), goat anti-ChAT (1:500, Millipore), mouse anti-PAX6 (1:200, Developmental Studies Hybridoma Bank). Secondary antibodies: Alexa Fluor-488 donkey anti-rabbit (1:500, Life Technologies), Alexa Fluor-555 donkey anti-goat (1:500, Life Technologies), Alexa-Fluor-555 donkey anti-mouse (1:500, Life Technologies).

Cai X., Huang H., Kuzirian M.S., Snyder L.M., Matsushita M., Lee M.C., Ferguson C., Homanics G.E., Barth A.L, & Ross S.E. (2015). Generation of a KOR-Cre Knockin Mouse Strain to Study Cells Involved in Kappa Opioid Signaling. Genesis (New York, N.Y. : 2000), 54(1), 29-37.

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Cerebellar Immunohistochemistry in Mice

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Mice were perfused and fixed with 4% paraformaldehyde for 2 hours at 4 °C, rinsed and sectioned on a vibratome (150 µm). Immunohistochemistry of floating parasagittal cerebellar sections was carried out essentially as described^{43 (link)}. The primary antibodies used were: rabbit anti-calbindin (Swant at 1:2500), anti-parvalbumin (Swant at 1:2000), rabbit anti-calretinin (Swant at 1:2000), chicken anti-βGal (AVES labs at 1:2000), chicken anti-GFP (AVES labs at 1:1000), rabbit anti-GFAP (abcam at 1:1000), guinea pig anti-vGlut2 (Millipore at 1:1000), rabbit anti-α6 (Millipore at 1:1000, discontinued), Mouse anti-GAD67 (Millipore at 1:500), rat anti-L1 (Millipore at 1:500) and mouse anti-pax6 (Developmental Studies Hybridoma Bank at 1:200). Sections were then washed in 1 × PBS and incubated with secondary antibodies and TOPRO-3 (Molecular Probe at 1:600 and 1:2000, respectively). Sections were washed in PBS and mounted using vectorshield hard-set fluorescence mounting medium (Vector laboratories). Confocal fluorescence images were taken using a Leica SPE II microscope. Area and length were measured using ImageJ. For cell counts, the ImageJ cell counter plugin was used⁴⁴.

Estep J.A., Wong W., Wong Y.C., Loui B.M, & Riccomagno M.M. (2018). The RacGAP β-Chimaerin is essential for cerebellar granule cell migration. Scientific Reports, 8, 680.

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Immunofluorescence Labeling of Neural Markers

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Slides were rehydrated in PBS then blocked with 10% goat serum, 3% BSA, 0,4% Triton X-100 in PBS for one hour. Primary antibodies were incubated overnight 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) 1:250 (Cell signaling #9101), rabbit anti-SOX2 1:500 (AB5603 Merck Millipore), mouse anti-TUJ1 1:500 (801202 Biolegend), rat anti-pHH3 1: 250 (S28, Abcam ab10543), rabbit anti-cleavedcaspase 3 1:500 (Asp175, CST 9661), mouse anti-PAX6 1:500 (Developmental Studies Hybridoma Bank), Phalloidin AlexaFluor 568 1:40 (Thermofisher). The secondary antibodies used were: anti-chicken, anti-rabbit, anti-mouse or anti-rat with conjugated fluorochromes (Alexa Fluor 488, 568 or 647; ThermoFisher) at 1:500. Sections were incubated for one hour in the blocking solution containing Hoechst dye (1:1000). Slides were washed, mounted (Thermo Scientific Shandon Immu-Mount) and imaged with a Zeiss microscope Z1 Apotome or a confocal LSM 780.

Wilmerding A., Bouteille L., Caruso N., Bidaut G., Etchevers H., Graba Y., & Delfini M. (2021). Sustained experimental activation of FGF8/ERK in the developing chicken spinal cord reproducibly models early events in ERK-mediated tumorigenesis.

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