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 7 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 Alexa Fluor® 488 Mouse anti-Human Pax-6 (Cat. No. 561664) and PerCP-Cy™5.5 Mouse anti-Human Sox1 (Cat. No. 561549) antibodies. The plot was derived from gated events based on light scattering characteristics for the neural induction. Flow cytometry was performed on a BD™ LSR II flow cytometry system. RIGHT: Immunofluorescent staining of Pax-6 in human embryonic stem (ES) cell-derived rosettes. H9 human ES cells (WiCell Madison, WI) grown on irradiated mouse embryonic fibroblasts were differentiated towards a neural stem cell lineage using a combination of 250 ng/ml human recombinant noggin (R&D systems) and 600 nM dorsomorphin (Sigma- Aldrich). The cells were fixed with BD CytofixT Fixation Buffer (Cat. No. 554655), permeabilized with 0.1% Triton™ X-100 Buffer, and stained with Alexa Fluor® 488 Mouse anti-Human Pax-6 (pseudo-colored green). Counter-staining was with Hoechst 33342 (pseudo-colored blue). The images were captured on a BD Pathway™ 435 Cell Analyzer and merged using BD Attovision™ Software.
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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 7 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 Alexa Fluor® 488 Mouse anti-Human Pax-6 (Cat. No. 561664) and PerCP-Cy™5.5 Mouse anti-Human Sox1 (Cat. No. 561549) antibodies. The plot was derived from gated events based on light scattering characteristics for the neural induction. Flow cytometry was performed on a BD™ LSR II flow cytometry system. RIGHT: Immunofluorescent staining of Pax-6 in human embryonic stem (ES) cell-derived rosettes. H9 human ES cells (WiCell Madison, WI) grown on irradiated mouse embryonic fibroblasts were differentiated towards a neural stem cell lineage using a combination of 250 ng/ml human recombinant noggin (R&D systems) and 600 nM dorsomorphin (Sigma- Aldrich). The cells were fixed with BD CytofixT Fixation Buffer (Cat. No. 554655), permeabilized with 0.1% Triton™ X-100 Buffer, and stained with Alexa Fluor® 488 Mouse anti-Human Pax-6 (pseudo-colored green). Counter-staining was with Hoechst 33342 (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 (Tested During Development)
5 µl
5080
AB_10895587
Aqueous buffered solution containing BSA, protein stabilizer, and ≤0.09% sodium azide.
RUO
Preparation And Storage
Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze. The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography. The antibody was conjugated to Alexa Fluor® 488 under optimum conditions, and unreacted Alexa Fluor® 488 was removed.
Recommended Assay Procedures
For Bioimaging procedures, please refer to the protocols under "Cellular Imaging" at our website: http://www.bdbiosciences.com/us/s/resources
Product Notices
- This reagent has been pre-diluted for use at the recommended Volume per Test. We typically use 1 × 10^6 cells in a 100-µl experimental sample (a test).
- An isotype control should be used at the same concentration as the antibody of interest.
- Source of all serum proteins is from USDA inspected abattoirs located in the United States.
- 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.
- Alexa Fluor® 488 fluorochrome emission is collected at the same instrument settings as for fluorescein isothiocyanate (FITC).
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- The Alexa Fluor®, Pacific Blue™, and Cascade Blue® dye antibody conjugates in this product are sold under license from Molecular Probes, Inc. for research use only, excluding use in combination with microarrays, or as analyte specific reagents. The Alexa Fluor® dyes (except for Alexa Fluor® 430), Pacific Blue™ dye, and Cascade Blue® dye are covered by pending and issued patents.
- Triton is a trademark of the Dow Chemical Company.
- mTESR™1 is a trademark of StemCell Technologies.
- Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
- Cy is a trademark of GE Healthcare.
- This reagent has been pre-diluted for use at the recommended Volume per Test when following the Recommended Assay Procedure. A Test is typically ~10,000 cells cultured in a well of a 96-well imaging plate.
- This antibody has been developed and certified for the bioimaging application. However, a routine bioimaging test is not performed on every lot. Researchers are encouraged to titrate the reagent for optimal performance.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
Companion Products
Stain Buffer (FBS) RUO
Size
500 mL
Cat No.
554656
Fixation Buffer RUO
Size
100 mL
Cat No.
554655
Perm Buffer III RUO
Size
125 mL
Cat No.
558050
Alexa Fluor®488 Mouse IgG2a, κ Isotype control RUO
Size
50 Tests
Cat No.
558055
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PerCP-Cy™5.5 Mouse anti-Human Sox1 RUO
Size
50 Tests
Cat No.
561549
561664 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.
561664 Rev. 2
Format Details
Alexa Fluor™ 488
Alexa Fluor™ 488 Dye is part of the BD blue family of dyes. This is a small organic fluorochrome with an excitation maximum (Ex Max) at 494-nm and an emission maximum (Em Max) at 517-nm. Alexa Fluor™ 488 is designed to be excited by the Blue laser (488 nm) and detected using an optical filter centered near 520-nm (e.g., a 530/30-nm bandpass filter). Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
Alexa Fluor™ 488
Blue 488 nm
494 nm
517 nm
561664 Rev.2
Citations & References
View product citations for antibody "561664" on CiteAb
Development References (4)
-
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
-
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). View Reference
-
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
561664 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.