Alexa Fluor® 647 Mouse Anti-Nestin
Clone 25/NESTIN (RUO)
- Brand BD Pharmingen™
- Vol. Per Test 5 µl
- Isotype Mouse IgG1, κ
- Reactivity Rat (QC Testing) Human (Tested in Development)
Bioimaging (Routinely Tested)
- Immunogen Rat Nestin aa. 402-604 Recombinant Protein
- Storage Buffer Aqueous buffered solution containing BSA, protein stabilizer, and ≤0.09% sodium azide.
- Regulatory Status RUO
Regulatory Status Legend
The cytoskeleton consists primarily of core structural proteins that include microfilaments, microtubules, and intermediate filaments (IFs). IFs contain more than 50 distinct proteins that are organized into six different subtypes: Type I/II keratins expressed in epithelia, type III vimentin/desmin, type IV neurofilament proteins, type V nuclear lamins, and type VI nestin expressed primarily in embryonic cells. Nestin has a conserved core region (amino acids 7 to 314), which contains an α helical domain that is involved in coiled-coil assembly of IFs. The C-terminal region of nestin is similar to type IV IFs, since it contains highly charged amino acids, many glutamate residues, and an 11 amino acid repeat motif. Nestin is expressed in the cerebrum during embryonic development, in the cerebellum during early postnatal development, and in dermatomal cells and myoblasts during myogenesis. In vitro, nestin forms homodimers and homotetramers, but not IFs, and can co-assemble with type III vimentin and type IV internexin proteins. Thus, nestin is a core IF protein that is essential for proper cytoskeletal formation during neurogenesis and myogenesis.
Alexa Fluor® 647 conjugates are highly photostable and remain fluorescent over a broad pH range. The excitation and emission maxima are nearly identical to those of APC. However, APC tends to be brighter while Alexa Fluor® 647 is more optimal for intracellular applications. This fluorochrome exhibits uncommon photostability, making it an ideal choice for use in fluorescence microscopy. Due to nearly identical excitation and emission properties but different spillover characteristics, APC and Alexa Fluor® 647 cannot be used simultaneously.
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Preparation and Storage
The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography.
The antibody was conjugated to Alexa Fluor® 647 under optimum conditions, and unreacted Alexa Fluor® 647 was removed.
Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze.
- Please refer to www.bdbiosciences.com/pharmingen/protocols for technical protocols.
- 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.
- 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.
- Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
- Triton is a trademark of the Dow Chemical Company.
- 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.
For more information, please refer to: http://www.bdbiosciences.com/pharmingen/protocols/Bioimaging_Certified.shtml or htpp://www.bdbiosciences.com/bioimaging/reagents.
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 μl of a 2 μg/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 855 620/60 700/75 660 LP
BD Pathway 435 628/40 690/40 FF660