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REACTIVITY SENSITIVITY MW (kDa) Isotype
H Endogenous Rabbit IgG
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Immunohistochemistry (Paraffin)

Immunohistochemical analysis of paraffin-embedded human Crohn's disease of the colon using CXCR4 (D4Z7W) Rabbit mAb.

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Immunohistochemistry (Paraffin)

Immunohistochemical analysis of paraffin-embedded Jurkat (left) and 293T (right) cell pellets using CXCR4 (D4Z7W) Rabbit mAb.

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Immunohistochemistry (Paraffin)

Immunohistochemical analysis of paraffin-embedded human tonsil using CXCR4 (D4Z7W) Rabbit mAb.

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Flow Cytometry

Flow cytometric analysis of K-562 cells (blue) and Daudi cells (green) using CXCR4 (D4Z7W) Rabbit mAb. Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.

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Flow Cytometry

Flow cytometric analysis of human peripheral blood mononucler cells using CXCR4 (D4Z7W) Rabbit mAb (right) co-stained with anti-human CD3-APC antibody compared to concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (left). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.

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Immunohistochemistry (Paraffin)

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

  1. Xylene.
  2. Ethanol, anhydrous denatured, histological grade (100% and 95%).
  3. Deionized water (dH2O).
  4. Hematoxylin (optional).
  5. Wash Buffer:
    1. 1X Tris Buffered Saline with Tween® 20 (TBST): To prepare 1L 1X TBST add 100 ml 10X Tris Buffered Saline with Tween® 20 (#9997) to 900 ml dH20, mix.
  6. SignalStain® Antibody Diluent (#8112).
  7. 1X Citrate Unmasking Solution: To prepare 250 mL of 1X citrate unmasking solution, dilute 25 ml of SignalStain® Citrate Unmasking Solution (10X) (#14746) with 225 mL of dH2O.
  8. 3% Hydrogen Peroxide: To prepare 100 ml, add 10 ml 30% H2O2 to 90 ml dH2O.
  9. Blocking Solution: TBST/5% Normal Goat Serum or 1X Animal-Free Blocking Solution.
    1. TBST/5% Normal Goat Serum: to 5 ml 1X TBST, add 250 µl Normal Goat Serum (#5425).
    2. 1X Animal-Free Blocking Solution: to 4 mL of dH2O add 1 ml of Animal-Free Blocking Solution (5X) (#15019).
  10. Detection System: SignalStain® Boost IHC Detection Reagents (HRP, Rabbit #8114).
  11. Substrate: SignalStain® DAB Substrate Kit (#8059).
  12. Hematoxylin: Hematoxylin (#14166).
  13. Mounting Medium: SignalStain® Mounting Medium (#14177).

B. Deparaffinization/Rehydration

NOTE: Do not allow slides to dry at any time during this procedure.

  1. Deparaffinize/hydrate sections:
    1. Incubate sections in three washes of xylene for 5 min each.
    2. Incubate sections in two washes of 100% ethanol for 10 min each.
    3. Incubate sections in two washes of 95% ethanol for 10 min each.
  2. Wash sections two times in dH2O for 5 min each.

C. Antigen Unmasking

For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; follow with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min.

D. Staining

  1. Wash sections in dH2O three times for 5 min each.
  2. Incubate sections in 3% hydrogen peroxide for 10 min.
  3. Wash sections in dH2O two times for 5 min each.
  4. Wash sections in wash buffer for 5 min.
  5. Block each section with 100–400 µl of preferred blocking solution for 1 hr at room temperature.
  6. Remove blocking solution and add 100–400 µl primary antibody diluted in SignalStain® Antibody Diluent (#8112) to each section. Incubate overnight at 4°C.
  7. Equilibrate SignalStain® Boost Detection Reagent (HRP, Rabbit #8114) to room temperature.
  8. Remove antibody solution and wash sections with wash buffer three times for 5 min each.
  9. Cover section with 1–3 drops SignalStain® Boost Detection Reagent (HRP, Rabbit #8114) as needed. Incubate in a humidified chamber for 30 min at room temperature.
  10. Wash sections three times with wash buffer for 5 min each.
  11. Add 1 drop (30 µl) SignalStain® DAB Chromogen Concentrate to 1 ml SignalStain® DAB Diluent and mix well before use.
  12. Apply 100–400 µl SignalStain® DAB to each section and monitor closely. 1–10 min generally provides an acceptable staining intensity.
  13. Immerse slides in dH2O.
  14. If desired, counterstain sections with hematoxylin (#14166).
  15. Wash sections in dH2O two times for 5 min each.
  16. Dehydrate sections:
    1. Incubate sections in 95% ethanol two times for 10 sec each.
    2. Repeat in 100% ethanol, incubating sections two times for 10 sec each.
    3. Repeat in xylene, incubating sections two times for 10 sec each.
  17. Mount sections with coverslips and mounting medium (#14177).

posted February 2010

revised March 2016

Protocol Id: 283
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Flow Cytometry

A. Solutions and Reagents

  1. 1X Phosphate Buffered Saline (PBS): Dissolve 8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4 and 0.24 g KH2PO4 in 800 mL distilled water (dH2O). Adjust the pH to 7.4 with HCl and the volume to 1 liter. Store at room temperature.
  2. Formaldehyde (methanol free).
  3. Incubation Buffer: Dissolve 0.5 g bovine serum albumin (BSA) in 100mL 1X PBS. Store at 4°C.

B. Fixation

  1. Collect cells by centrifugation and aspirate supernatant.
  2. Resuspend cells briefly in 0.5-1 ml PBS. Add formaldehyde to a final concentration of 2-4% formaldehyde.
  3. Fix for 10 minutes at 37°C.
  4. Add 5 ml PBS and rinse by centrifugation.
  5. Resuspend cells in 5 ml PBS.
  6. Proceed with staining or store cells at +4°C in PBS with 0.1% sodium azide.

C. Staining Using Unlabeled Primary and Conjugated Secondary Antibodies

NOTE: Allow species matched IgG for polyclonal antibodies. Count cells using a hemacytometer or alternative method.

  1. Aliquot 0.5-1x106 cells into each assay tube (by volume).
  2. Add 2-3 ml Incubation Buffer to each tube and rinse by centrifugation. Repeat.
  3. Resuspend cells in 100 μl Incubation Buffer per assay tube.
  4. Block in Incubation Buffer for 10 minutes at room temperature.
  5. Add the primary antibody at the appropriate dilution to the assay tubes (see individual antibody data sheet for the appropriate dilution).
  6. Incubate for 30-60 minutes at room temperature.
  7. Rinse as before in Incubation Buffer by centrifugation.
  8. Resuspend cells in fluorochrome-conjugated secondary antibody*, diluted in Incubation Buffer according to the manufacturer’s recommendations.
  9. Incubate for 30 minutes at room temperature.
  10. Rinse as before in Incubation Buffer by centrifugation.
  11. Resuspend cells in 0.5 ml PBS and analyze on flow cytometer.

*Recommended Secondary Antibodies:

Anti-Rabbit

posted January 2009

Protocol Id: 133

Product Usage Information

Application Dilutions
Immunohistochemistry (Paraffin) 1:800
Flow Cytometry 1:50

Storage: Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.

Specificity / Sensitivity

CXCR4 (D4Z7W) Rabbit mAb recognizes endogenous levels of total CXCR4 protein.


Species Reactivity: Human

Source / Purification

Monoclonal antibody is produced by immunizing animals with recombinant protein specific to the extracellular domains of human CXCR4 protein.

CXCR4 is a chemokine receptor that belongs to the G protein-coupled receptor family. It is activated by a small cytokine, CXCL12, also known as stromal cell derived factor 1 (SDF-1) (1). The main function of CXCR4 is the mediation of the homing of progenitor cells in the bone marrow and their recruitment to sites of injury (2). More recently, CXCR4 has been studied, as a potential therapeutic target, in the context of autoimmune diseases (3) as well as cancer, as the receptor is involved in the regulation of migration, proliferation, and survival of cancer cells (4).


1.  Blanchet, X. et al. (2012) Front Immunol 3, 175.

2.  Döring, Y. et al. (2014) Front Physiol 5, 212.

3.  Debnath, B. et al. (2013) Theranostics 3, 47-75.

4.  Teicher, B.A. and Fricker, S.P. (2010) Clin Cancer Res 16, 2927-31.


Entrez-Gene Id 7852
Swiss-Prot Acc. P61073


For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
SignalStain is a trademark of Cell Signaling Technology, Inc.
XP is a registered trademark of Cell Signaling Technology, Inc.
Alexa Fluor is a registered trademark of Life Technologies Corporation.

97680
CXCR4 (D4Z7W) Rabbit mAb