Rabbit anti sox2

Manufactured by Thermo Fisher Scientific

Sourced in United States, Germany

Rabbit anti-Sox2 is a primary antibody that recognizes the Sox2 (SRY-box transcription factor 2) protein. Sox2 is a key transcription factor involved in the regulation of embryonic development and the maintenance of stem cell pluripotency. This antibody can be used to detect and quantify Sox2 expression in various cell and tissue samples through techniques such as Western blotting, immunohistochemistry, and immunocytochemistry.

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Anti-SOX2 (5 citations)

7 protocols using rabbit anti sox2

Immunocytochemistry of Neural Progenitor Cells

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Neurospheres were placed into poly-D-lysine-coated glass-bottom dishes (Mattek) and allowed to attach for 6 hours while in a thin film of SCM, DMEM with 10% fetal bovine serum (FBS) and P/S (SM), or DMEM with B27M (Life Technologies, Grand Island, NY, USA) and P/S (B27M). After the neurospheres attached, 2 ml of medium (SCM, B27M or SM) was added to prevent loss of neurospheres. Neurospheres were fixed in 100% methanol for 10 minutes and standard immunocytochemistry was performed. Immunofluorescence staining was used to identify neural stem progenitor cells, neural progenitor cells, neurons, and astrocytes. Primary antibodies were used at the following dilutions: chicken anti-NeuN (Aves Labs, Tigard, OR, USA) 1:1000; chicken anti-Nestin (Aves Labs) 1:1000; rabbit anti-BetaIII-tubulin (Cell Signaling Technology, Danvers, MA, USA) 1:1000; rabbit anti-Musashi1 (Msi1, Cell Signaling Technology) 1:1000; rabbit anti-GFAP (Cell Signaling Technology) 1:1500; rabbit anti-SOX2 (Life Technologies) 1:500; cleaved caspase–3 (Cell Signaling Technology) 1:500. Samples were rinsed after overnight incubation at 4°C, and were incubated for 2 hours with appropriate Alexa488 and 458-conjugated secondary antibody (Life Technologies). Confocal microscopy of spheres was performed as mentioned in our previous study [35 (link)].

Malik A., Kondratov R.V., Jamasbi R.J, & Geusz M.E. (2015). Circadian Clock Genes Are Essential for Normal Adult Neurogenesis, Differentiation, and Fate Determination. PLoS ONE, 10(10), e0139655.

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Immunostaining of Pluripotent Stem Cells

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E14 ES cells were fixed for 20 min with a chilled mix of 95% methanol/5% acetic acid that was maintained at −20°C, permeabilized with chilled PBS-Triton for 15 min, and blocked with PBS and 1% BSA for 30–60 min. Samples were incubated with primary antibodies rabbit anti-Sox2 (1:200; Life Technologies) and rabbit anti-Oct4 (1:500; Abcam, ab19857) in PBS and 1% BSA overnight at 4°C. Samples were washed twice in PBS and then incubated with Alexa 555 anti-rabbit (1:1000; Life Technologies, catalog no. 481400) in PBS and 1% BSA for 45–60 min followed by three washes with 0.1% PBS-Tween, incubation with 2 ng/mL DAPI, three washes with PBS and 0.1% Tween, and two washes with PBS.

Deluz C., Friman E.T., Strebinger D., Benke A., Raccaud M., Callegari A., Leleu M., Manley S, & Suter D.M. (2016). A role for mitotic bookmarking of SOX2 in pluripotency and differentiation. Genes & Development, 30(22), 2538-2550.

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Immunofluorescence Analysis of Stem Cells

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For immunofluorescence, TSC-patient-specific and unaffected control iPSCs, pNSCs, neurons, and astrocytes were fixed in 4% (w/v) paraformaldehyde at room temperature for 15 min, permeabilized for 15 min in PBS containing 0.5% (v/v) Triton X-100, blocked with 5% (w/v) BSA for 1 hr at room temperature, and incubated with primary antibodies overnight at 4°C in PBS containing 1% (w/v) BSA. Cells were then washed three times in PBS and incubated for 1 hr at room temperature with anti-rabbit or anti-mouse Alexa Fluor 488- and/or 555-conjugated secondary antibodies (Life Technologies). Cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole. The primary antibodies used were as follows: rabbit anti-OCT4 (Abcam, catalog no. ab19857), rabbit anti-SOX2 (Abcam, catalog no. ab97959), mouse anti-SSEA4 (Abcam, catalog no. ab16287), mouse anti-TRA-1-81 (Abcam, catalog no. ab16289), mouse anti-AFP (Abcam, catalog no. ab3980), rabbit anti-SMA (Abcam, catalog no. ab5694), mouse anti-NESTIN (Life Technologies, catalog no. A24353), goat anti-SOX1 (Life Technologies, catalog no. A24354), rabbit anti-SOX2 (Life Technologies, catalog no. A24354), rabbit anti-PAX6 (Life Technologies, catalog no. A24354), rabbit anti-β-III-TUBULIN (TUJ1; Abcam, catalog no. ab18207), and rabbit anti-GFAP (Abcam, catalog no. ab7260).

Li Y., Cao J., Chen M., Li J., Sun Y., Zhang Y., Zhu Y., Wang L, & Zhang C. (2017). Abnormal Neural Progenitor Cells Differentiated from Induced Pluripotent Stem Cells Partially Mimicked Development of TSC2 Neurological Abnormalities. Stem Cell Reports, 8(4), 883-893.

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Immunofluorescence Staining Protocol

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The following antibodies were used: rabbit anti-GFAP (DAKO Agilent, Santa Clara, CA, Z0334), rabbit anti-caspase-6 (Cell Signaling Technologies, Danvers, MA, 9762), rabbit anti-Caspase-6 (abcam, Cambridge, UK, ab185645), rabbit anti-D225 (Chen et al., 2013 (link)), mouse anti-GFAP (Sigma, GA5), mouse anti-pSer13-GFAP (KT13 [Sekimata et al., 1996 (link)]), mouse anti-pan Actin, mouse anti-Tra-1–60, mouse anti-SSEA4, rabbit anti-Oct4, rabbit anti-Sox2, and Alexa 488- and Alexa 594-congujated goat anti mouse or rabbit antibodies (Thermo Fisher Scientific, Waltham, MA).

Battaglia R.A., Beltran A.S., Delic S., Dumitru R., Robinson J.A., Kabiraj P., Herring L.E., Madden V.J., Ravinder N., Willems E., Newman R.A., Quinlan R.A., Goldman J.E., Perng M.D., Inagaki M, & Snider N.T. (2019). Site-specific phosphorylation and caspase cleavage of GFAP are new markers of Alexander disease severity. eLife, 8, e47789.

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Immunofluorescence Characterization of iPSCs and Embryoid Bodies

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iPSC colonies and embryoid bodies were fixed using 4% paraformaldehyde (PFA) and permeabilized with 0.1% Triton X-100 (Sigma-Aldrich, St. Quentin Fallavier, France) in PBS. Non-specific binding was blocked with 1% BSA (Sigma-Aldrich) and 10% donkey serum (Millipore, Saint Quentin en Yvelines, France). Primary antibodies were used at a 1:200 dilution in blocking solution and incubated overnight at 4°C: rabbit anti-NANOG (Abcam, Paris, France), mouse anti-OCT3/4 (Santa Cruz Biotechnology, Heidelberg, Germany), and rabbit anti-SOX2 (Thermo Fisher Scientific) for the iPSCs, and rabbit anti-GFAP (Dako, Les Ulis, France), mouse anti-SMA (Dako), and mouse anti-AFP (Sigma Aldrich) for the embryoid bodies. Fluorescence-conjugated secondary anti-mouse and anti-rabbit antibodies (Jackson ImmunoResearch Laboratories, Suffolk, UK) were used at a 1:500 dilution and incubated for 1 h at room temperature. Nuclei were stained with 0.2 μg/mL bisBenzimide (Sigma-Aldrich). Cells were imaged using a Zeiss ApoTome 2 Upright wide-field microscope.

Sanjurjo-Soriano C., Erkilic N., Baux D., Mamaeva D., Hamel C.P., Meunier I., Roux A.F, & Kalatzis V. (2019). Genome Editing in Patient iPSCs Corrects the Most Prevalent USH2A Mutations and Reveals Intriguing Mutant mRNA Expression Profiles. Molecular Therapy. Methods & Clinical Development, 17, 156-173.

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Comprehensive Immunofluorescence and Western Blot Antibody Protocol

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The following primary antibodies and concentrations were used in this study: rabbit anti-GFAP (DAKO, Agilent, clone Z0334; IF 1:500, WB 1:10,000), mouse anti-GFAP (Sigma, clone GA5; IF 1:300), mouse anti-Gigaxonin (Santa Cruz Biotechnology, F3, WB 1:200), rabbit anti-Vimentin (Cell-Signaling Technology, D21H3, IF 1:100), mouse anti-Vimentin (Thermo Fisher Scientific, V9, WB 1:1000), mouse anti-Keratin 8 (Thermo Fisher Scientific, TS1, IF 1:100), rat anti-K8 (Developmental Studies Hybridoma Bank, Troma I, WB 1:5000), rabbit anti-Lamin A/C (Santa Cruz Biotechnology), rabbit anti-Lamin B1 (Abcam ab16048, IF 1:10,000, WB 1:10,000), mouse anti-Nestin (Thermo Fisher Scientific, 10C2, IF 1:200), mouse anti-NF-M/H (clone RMdO-20; IF 1:100), mouse anti-Tra-1-60 (Thermo Fisher Scientific, 41-1000, IF 1:300), mouse anti-Tra-1-81 (Thermo Fisher Scientific, 41-1100, IF 1:300), rabbit anti-Oct4 (Abcam, ab19857, IF 1:40), and rabbit anti-Sox2 (Thermo Fisher Scientific, 48-1400, IF 1:125). The following secondary antibodies and concentrations were used: Alexa 488- and Alexa 594-conjugated goat-, anti-mouse-, and rabbit-antibodies (Thermo Fisher Scientific, IF 1:500), and peroxidase-conjugated goat-, anti-mouse-, and rabbit-antibodies (Sigma, WB 1:5000).

Battaglia R.A., Faridounnia M., Beltran A., Robinson J., Kinghorn K., Ezzell J.A., Bharucha-Goebel D., Bönnemann C.G., Hooper J.E., Opal P., Bouldin T.W., Armao D, & Snider N.T. (2023). Intermediate filament dysregulation in astrocytes in the human disease model of KLHL16 mutation in giant axonal neuropathy (GAN). Molecular Biology of the Cell, 34(12), ar121.

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Comprehensive Antibody Panel for Cellular Characterization

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The following primary antibodies and concentrations were used in this study: rabbit anti-GFAP (DAKO, Agilent, clone Z0334; IF 1:500, WB 1:10,000), mouse anti-GFAP (Sigma, clone GA5; IF 1:300), mouse anti-pSer13-GFAP (gift from Dr. Masaki Inagaki, clone KT13, IF 1:20), mouse anti-Gigaxonin (Santa Cruz Biotechnology, F3, WB 1:200), rabbit anti-Vimentin (Cell Signaling Technology, D21H3, IF 1:100), mouse anti-Vimentin (Thermo Fisher Scientific, V9, WB 1:1000), mouse anti-Keratin 8 (Thermo Fisher Scientific, TS1, IF 1:100), rat anti-K8 (Developmental Studies Hybridoma Bank, Troma I, WB 1:5000), rabbit anti-Lamin A/C (Santa Cruz Biotechnology), rabbit anti-Lamin B1 (Abcam ab16048, IF 1:10,000, WB 1:10,000), mouse anti-Nestin (Thermo Fisher Scientific, 10C2, IF 1:200), mouse anti-Tra-1–60 (Thermo Fisher Scientific, 41–1000, IF 1:300), mouse anti-Tra-1–81 (Thermo Fisher Scientific, 41–1100, IF 1:300), rabbit anti-Oct4 (Abcam, ab19857, IF 1:40), and rabbit anti-Sox2 (Thermo Fisher Scientific, 48–1400, IF 1:125). The following secondary antibodies and concentrations were used: Alexa 488- and Alexa 594-conjugated goat anti mouse and rabbit antibodies (Thermo Fisher Scientific, IF 1:500), and peroxidase-conjugated goat anti-mouse and rabbit antibodies (Sigma, WB 1:5000).

Battaglia R., Faridounnia M., Beltran A., Robinson J., Kinghorn K., Ezzell J.A., Bharucha-Goebel D., Bonnemann C., Hooper J.E., Opal P., Bouldin T.W., Armao D, & Snider N. (2023). Intermediate filament dysregulation and astrocytopathy in the human disease model of KLHL16 mutation in giant axonal neuropathy (GAN). bioRxiv.

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