LEFT: Intracellular staining of Pax-6 in neural induction of human embryonic stem (ES) cells. H9 human ES cells (WiCell, Madison, WI) were cultured in mTeSR® (Stem Cell Technologies) on plates coated with BD Matrigel™ hESC-qualified Matrix (Cat. No. 354277). Embryoid bodies (EB) were made and cultured in medium containing Knockout™ Serum Replacement (Life Technologies) without bFGF for 24 hours and then in medium containing 250 ng/ml human recombinant noggin (R&D Systems) and 10 μM SB 431542 (Tocris) for 4 more days. The EB were then plated on BD Matrigel-coated plates and grown in medium with ITS supplement (Sigma-Aldrich), noggin, and SB 431542. After growth for 21 days, the cells were collected, fixed with BD Cytofix™ Fixation Buffer (Cat. No. 554655), and permeabilized with BD Phosflow™ Perm Buffer III (Cat. No. 558050). The cells were then stained with either Purified Mouse anti-Human Pax-6 antibody (solid line histrogram) or Purified Mouse IgG2a, κ isotype control (dashed line, Cat. No. 554126). The second-step reagent was PE Goat Anti-Mouse Ig (Multiple Adsorption, Cat. No. 550589). Flow cytometry was performed on a BD LSR™ II flow cytometry system. RIGHT: Immunofluorescent staining of Pax-6 in human embryonic stem cell-derived rosettes. H9 human ES cells (WiCell, Madison, WI) passage 54 cultured in mTeSR™1 medium (StemCell Technologies) on BD Matrigel™ hESC-qualified Matrix (Cat. No. 354277) were differentiated towards a neural stem cell lineage. Cells were fixed with BD Cytofix™ Fixation Buffer (Cat. No. 554655), permeabilized with 0.1% Triton™ X-100 [Perm Buffer III is also suitable], and stained with Purified Mouse anti-Human Pax-6 (pseudo-colored green) at 2.5 µg/mL. The second-step reagent was Alexa Fluor® 488 goat anti-mouse Ig (Life Technologies) and counter-staining was with Hoechst 33342 (Sigma-Aldrich, pseudo-colored blue). The images were captured on a BD Pathway™ 435 Cell Analyzer and merged using BD Attovision™ Software.
LEFT: Intracellular staining of Pax-6 in neural induction of human embryonic stem (ES) cells. H9 human ES cells (WiCell, Madison, WI) were cultured in mTeSR® (Stem Cell Technologies) on plates coated with BD Matrigel™ hESC-qualified Matrix (Cat. No. 354277). Embryoid bodies (EB) were made and cultured in medium containing Knockout™ Serum Replacement (Life Technologies) without bFGF for 24 hours and then in medium containing 250 ng/ml human recombinant noggin (R&D Systems) and 10 μM SB 431542 (Tocris) for 4 more days. The EB were then plated on BD Matrigel-coated plates and grown in medium with ITS supplement (Sigma-Aldrich), noggin, and SB 431542. After growth for 21 days, the cells were collected, fixed with BD Cytofix™ Fixation Buffer (Cat. No. 554655), and permeabilized with BD Phosflow™ Perm Buffer III (Cat. No. 558050). The cells were then stained with either Purified Mouse anti-Human Pax-6 antibody (solid line histrogram) or Purified Mouse IgG2a, κ isotype control (dashed line, Cat. No. 554126). The second-step reagent was PE Goat Anti-Mouse Ig (Multiple Adsorption, Cat. No. 550589). Flow cytometry was performed on a BD LSR™ II flow cytometry system. RIGHT: Immunofluorescent staining of Pax-6 in human embryonic stem cell-derived rosettes. H9 human ES cells (WiCell, Madison, WI) passage 54 cultured in mTeSR™1 medium (StemCell Technologies) on BD Matrigel™ hESC-qualified Matrix (Cat. No. 354277) were differentiated towards a neural stem cell lineage. Cells were fixed with BD Cytofix™ Fixation Buffer (Cat. No. 554655), permeabilized with 0.1% Triton™ X-100 [Perm Buffer III is also suitable], and stained with Purified Mouse anti-Human Pax-6 (pseudo-colored green) at 2.5 µg/mL. The second-step reagent was Alexa Fluor® 488 goat anti-mouse Ig (Life Technologies) and counter-staining was with Hoechst 33342 (Sigma-Aldrich, pseudo-colored blue). The images were captured on a BD Pathway™ 435 Cell Analyzer and merged using BD Attovision™ Software.
Product Details
BD Pharmingen™
Oculorhombin, Aniridia type II protein, PAX6, AN2
Human (QC Testing)
Mouse BALB/c IgG2a, κ
Human Pax-6 aa 406-422 Peptide
Intracellular staining (flow cytometry) (Routinely Tested), Bioimaging, Immunofluorescence (Tested During Development), Western blot (Not Recommended)
46-48 kDa
0.5 mg/ml
5080
AB_10715442
Aqueous buffered solution containing ≤0.09% sodium azide.
RUO
Preparation And Storage
Store undiluted at 4°C. The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography.
Product Notices
- Since applications vary, each investigator should titrate the reagent to obtain optimal results.
- An isotype control should be used at the same concentration as the antibody of interest.
- mTESR™1 is a trademark of StemCell Technologies.
- Alexa Fluor® is a registered trademark of Life Technologies Corporation.
- Triton is a trademark of the Dow Chemical Company.
- Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
Companion Products
Stain Buffer (FBS) RUO
Size
500 mL
Cat No.
554656
Perm Buffer III RUO
Size
125 mL
Cat No.
558050
Fixation Buffer RUO
Size
100 mL
Cat No.
554655
Purified Mouse IgG2a, κ Isotype Control RUO
Size
0.1 mg
Cat No.
554126
PE Goat Anti-Mouse Ig (Multiple Adsorption) RUO
Size
0.2 mg
Cat No.
550589
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561462 Rev. 2
Antibody Details
O18-1330
Pax-6 is a member of the paired box (pax) gene family whose protein products are transcription factors involved in development. Pax family members share a highly conserved DNA binding domain that contains six alpha helices (paired domain) and a homeo box domain. Pax-6 has important roles in the development of the eye, nose, central nervous system, and pancreas. Defects in Pax-6 are responsible for various eye malformations including aniridia and Peters anomaly.
The O18-1330 monoclonal antibody reacts with human Pax-6. Because the Pax-6 protein sequence is highly conserved among vertebrate species, cross-reactivity with other species is possible.
561462 Rev. 2
Format Details
Purified
Tissue culture supernatant is purified by either protein A/G or affinity purification methods. Both methods yield antibody in solution that is free of most other soluble proteins, lipids, etc. This format provides pure antibody that is suitable for a number of downstream applications including: secondary labeling for flow cytometry or microscopy, ELISA, Western blot, etc.
Purified
561462 Rev.2
Citations & References
Development References (4)
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Cerf ME. Transcription factors regulating beta-cell function. Eur J Endocrinol. 2006; 155(5):671-679. (Biology). View Reference
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Chambers SM, Fasano CA, Papapetrou EP, Tomishima M, Sadelain M, Studer L. Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat Biotechnol. 2009; 27(3):275-280. (Methodology). View Reference
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Glaser T, Walton DS, Maas RL. Genomic structure, evolutionary conservation and aniridia mutations in the human PAX6 gene. Nat Genet. 1992; 2:232-239. (Biology: Stimulation). View Reference
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Osakada F, Jin ZB, Hirami Y, et al. In vitro differentiation of retinal cells from human pluripotent stem cells by small-molecule induction. J Cell Sci. 2009; 122:3169-3179. (Methodology). View Reference
561462 Rev. 2
Please refer to Support Documents for Quality Certificates
Global - Refer to manufacturer's instructions for use and related User Manuals and Technical data sheets before using this products as described
Comparisons, where applicable, are made against older BD Technology, manual methods or are general performance claims. Comparisons are not made against non-BD technologies, unless otherwise noted.
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
