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Alexa Fluor® 555 Mouse anti-Oct3/4
Alexa Fluor® 555 Mouse anti-Oct3/4

Immunofluorescent staining of human ES cell line.  H9 cells (WiCell, Madison, WI) were grown on mouse epithelial feeder cells in a BD Falcon™ 96-well Imaging Plate (Cat. No. 353219).  After overnight incubation, the cells were fixed, permeabilized with BD Perm/Wash™ Buffer (Cat No. 554723), stained with Alexa Fluor® 555 Mouse anti-Oct 3/4 (pseudo colored red), and counter stained with Hoechst 33342 (pseudo colored blue) according to the Recommended Assay Procedure.  The images were captured on a BD Pathway™ 435 High-Content Bioimager System with a 20x objective and merged using BD AttoVision™ software. This antibody also stained human EC NCCIT (ATCC, Cat. No. CRL-2073), mouse EC F9 (ATCC, Cat. No. CRL-1720), and mouse ES-E14TG2a (ATCC, Cat. No. CRL-1821) cells.  It also works with Triton™ X-100 and methanol permeabilization (see Recommended Assay Procedure).

Immunofluorescent staining of human ES cell line.  H9 cells (WiCell, Madison, WI) were grown on mouse epithelial feeder cells in a BD Falcon™ 96-well Imaging Plate (Cat. No. 353219).  After overnight incubation, the cells were fixed, permeabilized with BD Perm/Wash™ Buffer (Cat No. 554723), stained with Alexa Fluor® 555 Mouse anti-Oct 3/4 (pseudo colored red), and counter stained with Hoechst 33342 (pseudo colored blue) according to the Recommended Assay Procedure.  The images were captured on a BD Pathway™ 435 High-Content Bioimager System with a 20x objective and merged using BD AttoVision™ software. This antibody also stained human EC NCCIT (ATCC, Cat. No. CRL-2073), mouse EC F9 (ATCC, Cat. No. CRL-1720), and mouse ES-E14TG2a (ATCC, Cat. No. CRL-1821) cells.  It also works with Triton™ X-100 and methanol permeabilization (see Recommended Assay Procedure).

Product Details
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BD Pharmingen™
Oct3, OTF3, Oct4, OTF4, POU5F1
Human (QC Testing), Mouse (Tested in Development)
Mouse IgG1, κ
Mouse Oct3 aa. 252-372 Recombinant Protein
Bioimaging (Routinely Tested)
5 µl
AB_1645312
Aqueous buffered solution containing BSA, protein stabilizer, and ≤0.09% sodium azide.
RUO


Preparation And Storage

The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography. The antibody was conjugated to Alexa Fluor® 555 under optimum conditions, and unreacted Alexa Fluor® 555 was removed. Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze.

Recommended Assay Procedures

For more information, please refer to: Bioimaging: http://www.bdbiosciences.com/pharmingen/protocols/Bioimaging_Certified.shtml

Recommended Protocol for Bioimaging

1.        Seed the cells in appropriate culture medium at an appropriate cell density in a BD Falcon™ 96-well Imaging Plate (Cat. No. 353219), and

        culture overnight to 48 hours.

2.        Remove the culture medium from the wells, and wash (one to two times) with 100 µl of 1× PBS.

3.        Fix the cells by adding 100 µl of fresh 3.7% Formaldehyde in PBS or BD Cytofix™ fixation buffer (Cat. No. 554655) to each well and

        incubating for 10 minutes at room temperature (RT).

4.        Remove the fixative from the wells, and wash the wells (one to two times) with 100 µl of 1× PBS.

5.        Permeabilize the cells using either cold methanol (a), Triton™ X-100 (b), or Saponin (c):

                a.        Add 100 µl of -20°C 90% methanol or -20°C BD™ Phosflow Perm Buffer III (Cat. No. 558050) to each well and incubate for 5

                        minutes at RT.

                b.        Add 100 µl of 0.1% Triton™ X-100 to each well and incubate for 5 minutes at RT.

                c.        Add 100 µl of 1× Perm/Wash buffer (Cat. No. 554723) to each well and incubate for 15 to 30 minutes at RT.  Continue to use 1×

                        Perm/Wash buffer for all subsequent wash and dilutions steps.

6.        Remove the permeabilization buffer from the wells, and wash one to two times with 100 µl of appropriate buffer (either 1× PBS or 1×

        Perm/Wash buffer, see step 5.c.).

7.        Optional blocking step:  Remove the wash buffers, and block the cells by adding 100 µl of blocking buffer BD Pharmingen™ Stain Buffer

        (FBS) (Cat. No. 554656) or 3% FBS in appropriate dilution buffer to each well and incubating for 15 to 30 minutes at RT.

8.        Dilute the antibody to its optimal working concentration in appropriate dilution buffer.  Titrate purified (unconjugated) antibodies and

        second-step reagents to determine the optimal concentration.  If using a Bioimaging Certified antibody conjugate, dilute it 1:10.

9.        Add 50 µl of diluted antibody per well and incubate for 60 minutes at RT.  Incubate in the dark if using fluorescently labeled antibodies.

10.        Remove the antibody, and wash the wells three times with 100 µl of wash buffer.  An optional detergent wash (100 µl of 0.05% Tween in 1×

        PBS) can be included prior to the regular wash steps.

11.   If the antibody being used is fluorescently labeled, then move to step 12.  Otherwise, if using a purified unlabeled antibody, repeat steps 8 to

        10 with a fluorescently labeled second-step reagent to detect the purified antibody.

12.        After the final wash, counter-stain the nuclei by adding 100  l of a 2  g/ml solution of Hoechst 33342 (eg, Sigma-Aldrich Cat. No. B2261) in

        1× PBS to each well at least 15 minutes before imaging.

13.        View and analyze the cells on an appropriate imaging instrument.  Recommended filters for the BD Pathway™ instruments are:

        Instrument                        Excitation        Emission        Dichroic

        BD Pathway 855                548/20                570LP                Fura/FITC                

        BD Pathway 435                543/22                593/40                FF562

Product Notices

  1. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
  2. 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.
  3. 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.
  4. Triton is a trademark of the Dow Chemical Company.
  5. 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.
  6. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
  7. Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
560306 Rev. 1
Antibody Details
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40/Oct-3

Development of a multicellular organism from a single fertilized cell is regulated by the coordinated activity of DNA transcription factors.  Oct3/4, a member of the POU family of transcription factors, functions in pluripotent cells of early embryonic stem cell (ES) lines and embryonal carcinomas (EC).  Other members of the POU family include Oct1, Oct2, Pit-1, and unc-86.  The POU domain, a 150-amino acid region that determines binding specificity, is conserved among these proteins and consists of 3 subdomains: POU-specific A and B subdomains and a homeobox-like subdomain.  Oct3/4 is expressed in undifferentiated cells, but is lost as cells are induced to differentiate.  Oct3/4 is not expressed in adult tissues.  The interaction of Oct3/4 with SOX2, another embryonic transcription factor, produces an active complex that regulates expression of genes such as Nanog, UTF1, and FGF4.  Although Oct3/4 is specifically phosphorylated on serine residues, this modification is not required for DNA binding, but may affect its transactivation potential.  Thus, Oct3/4 is a transcription factor that plays an important role in determining early steps of embryogenesis and differentiation.

560306 Rev. 1
Format Details
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Alexa Fluor™ 555
Alexa Fluor™ 555
560306 Rev.1
Citations & References
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Development References (6)

  1. Nishimoto M, Fukushima A, Okuda A, Muramatsu M. The gene for the embryonic stem cell coactivator UTF1 carries a regulatory element which selectively interacts with a complex composed of Oct-3/4 and Sox-2. Mol Cell Biol. 1999; 19(8):5453-5465. (Biology). View Reference
  2. Okamoto K, Okazawa H, Okuda A, Sakai M, Muramatsu M, Hamada H. A novel octamer binding transcription factor is differentially expressed in mouse embryonic cells. Cell. 1990; 60(3):461-472. (Biology). View Reference
  3. Pan G, Thomson JA. Nanog and transcriptional networks in embryonic stem cell pluripotency. Cell Res. 2007; 17:42-49. (Biology). View Reference
  4. Rosfjord E, Scholtz B, Lewis R, Rizzino A. Phosphorylation and DNA binding of the octamer binding transcription factor Oct-3. Biochem Biophys Res Commun. 1995; 212(3):847-853. (Biology). View Reference
  5. Vigano MA, Staudt LM. Transcriptional activation by Oct-3: evidence for a specific role of the POU-specific domain in mediating functional interaction with Oct-1. Nucleic Acids Res. 1996; 24(11):2112-2118. (Biology). View Reference
  6. Yuan H, Corbi N, Basilico C, Dailey L. Developmental-specific activity of the FGF-4 enhancer requires the synergistic action of Sox2 and Oct-3. Genes Dev. 1995; 9(21):2635-2645. (Biology). View Reference
View All (6) View Less
560306 Rev. 1

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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.