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Sox2 (D6D9) XP® Rabbit mAb (Alexa Fluor® 555 Conjugate) #5179
Gallery: Sox2 (D6D9) XP® Rabbit mAb (Alexa Fluor® 555 Conjugate) #5179
A. Solutions and Reagents
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
- 20X Phosphate Buffered Saline (PBS): (9808) To prepare 1L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix. Adjust pH to 8.0.
- Formaldehyde: 16%, methanol free, Polysciences, Inc. (cat# 18814), use fresh and store opened vials at 4°C in dark, dilute in 1X PBS for use.
- Blocking Buffer (1X PBS / 5% normal goat serum (#5425) / 0.3% Triton™ X-100): To prepare 10 ml: add 0.5 ml normal goat serum and 0.5 ml 20X PBS to 9.0 ml dH2O, mix. While stirring, add 30 µl Triton™ X-100.
- Antibody Dilution Buffer: (1X PBS / 1% BSA / 0.3% Triton™ X-100): To prepare 10 ml, add 30 µl Triton™ X-100 to 10 ml 1X PBS. Mix well then add 0.1 g BSA (#9998), mix.
- Prolong® Gold AntiFade Reagent (#9071), Prolong® Gold AntiFade Reagent with DAPI (#8961).
B. Specimen Preparation - Cultured Cell Lines (IF-IC)
NOTE: Cells should be grown, treated, fixed and stained directly in multi-well plates, chamber slides or on coverslips.
Aspirate liquid, then cover cells to a depth of 2–3 mm with 4% formaldehyde diluted in 1X PBS.
NOTE: Formaldehyde is toxic, use only in a fume hood.
- Allow cells to fix for 15 min at room temperature.
- Aspirate fixative, rinse three times in 1X PBS for 5 min each.
- Proceed with Immunostaining (Section C).
NOTE: All subsequent incubations should be carried out at room temperature unless otherwise noted in a humid light-tight box or covered dish/plate to prevent drying and fluorochrome fading.
- Block specimen in Blocking Buffer for 60 min.
- While blocking, prepare primary antibody by diluting as indicated on datasheet in Antibody Dilution Buffer.
- Aspirate blocking solution, apply diluted primary antibody.
- Incubate overnight at 4°C.
- Rinse three times in 1X PBS for 5 min each.
- Coverslip slides with Prolong® Gold Antifade Reagent (#9071) or Prolong® Gold Antifade Reagent with DAPI (#8961).
- For best results, allow mountant to cure overnight at room temperature. For long-term storage, store slides flat at 4°C protected from light.
posted November 2006
revised November 2013
protocol id: 182
Sox2 (D6D9) XP® Rabbit mAb (Alexa Fluor® 555 Conjugate) detects endogenous levels of Sox2 protein.Species Reactivity: Human Species predicted to react based on 100% sequence homology: Monkey, Bovine, Dog, Horse
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to amino acids surrounding Gly179 of human Sox2.
This Cell Signaling Technology antibody is conjugated to Alexa Fluor® 555 fluorescent dye and tested in-house for immunofluorescent analysis in human cells. The antibody is expected to exhibit the same species cross-reactivity as the unconjugated Sox2 (D6D9) XP® Rabbit mAb #3579.
Embryonic stem cells are derived from the inner cell mass of the blastocyst and are unique in their pluripotent capacity and potential for self-renewal. Sox2 is one of a set of transcription factors that are crucial for the maintenance of pluripotency (1). Sox2, Oct-4, and Nanog cooperate in this network (1-3), and siRNA knockdown of either Sox2 or Oct-4 results in loss of pluripotency (4,5). Chromatin immunoprecipitation experiments have shown that Sox2 and Oct-4 bind to thousands of gene regulatory sites, highlighting the importance of these transcription factors in early embryonic development (6,7). It has recently been shown that Sox2 is amplified in lung and esophageal squamous cell tumors (8).
For Research Use Only. Not For Use In Diagnostic Procedures. Cell Signaling Technology® is a trademark of Cell Signaling Technology, Inc. XP® is a trademark of Cell Signaling Technology, Inc. The Alexa Fluor® dye antibody conjugates in this product are sold under license from Life Technologies Corporation for research use only, except for use in combination with DNA microarrays. The Alexa Fluor® dyes (except for Alexa Fluor® 430 dye) are covered by pending and issued patents. Alexa Fluor® is a registered trademark of Molecular Probes, Inc.