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REACTIVITY SENSITIVITY MW (kDa) Isotype
H Endogenous 110 Mouse IgG1
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Immunohistochemistry (Paraffin)

Immunohistochemical analysis of paraffin-embedded human capillary hemangioma using CD34 (ICO115) Mouse mAb.

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Flow Cytometry

Flow cytometric analysis of peripheral blood spiked with CD34-positive Stem-Trol™ Control Cells (Beckman Coulter) using CD34 (ICO-115) Mouse mAb.

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Immunohistochemistry (Paraffin)

A. Solutions and Reagents

  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. Antigen Unmasking Citrate: 10 mM Sodium Citrate Buffer: To prepare 1 L, add 2.94 g sodium citrate trisodium salt dihydrate (C6H5Na3O7•2H2O) to 1 L dH2O. Adjust pH to 6.0.
  7. 3% Hydrogen Peroxide: To prepare, add 10 ml 30% H2O2 to 90 ml dH2O.
  8. Blocking Solution: TBST/5% normal goat serum (#5425): to 5 ml 1X TBST add 250 µl normal goat serum.
  9. Detection System: VECTASTAIN® Elite ABC, including biotinylated secondary antibody (Vector Laboratories).
  10. Substrate: Vector® NovaRED™ (Vector Laboratories).

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 minutes each.
    2. Incubate sections in two washes of 100% ethanol for 10 minutes each.
    3. Incubate sections in two washes of 95% ethanol for 10 minutes each.
  2. Wash sections twice in dH2O for 5 minutes each.

C. Antigen Unmasking

For Citrate: Bring slides to a boil in 10 mM sodium citrate buffer, pH 6.0; maintain at a sub-boiling temperature for 10 min. Cool slides on bench top for 30 min.

D. Staining

  1. Wash sections in dH2O three times for 5 minutes each.
  2. Incubate sections in 3% hydrogen peroxide for 10 minutes.
  3. Wash sections in dH2O twice for 5 minutes each.
  4. Wash sections in wash buffer for 5 minutes.
  5. Block each section with 100-400 µl blocking solution for 1 hour at room temperature.
  6. Remove blocking solution and add 100-400 µl primary antibody diluted in blocking solution to each section. Incubate overnight at 4°C.
  7. Prepare ABC solution per manufacturer's recommendations.
  8. Remove primary antibody and wash section three times with wash buffer for 5 minutes each.
  9. Add 100-400 µl biotinylated secondary antibody, diluted in TBST per manufacturer’s recommendation, to each section. Incubate 30 minutes at room temperature.
  10. Remove secondary antibody solution and wash sections three times with wash buffer for 5 minutes each.
  11. Cover sections with 100-400 µl pre-mixed ABC solution as needed and incubate in a humidified chamber for 30 min at room temperature.
  12. Wash section three times with wash buffer for 5 min each.
  13. Prepare Vector® NovaRED™ per manufacturer's recommendations.
  14. Apply 100-400 µl substrate to each section and monitor closely. 5-15 minutes generally provides an acceptable staining intensity.
  15. If desired, counterstain sections in hematoxylin per manufacturer’s instructions.
  16. Wash sections in dH2O two times for 5 minutes each.
  17. Dehydrate sections:
    1. Incubate sections in 95% ethanol two times for 10 seconds each.
    2. Repeat in 100% ethanol, incubating sections two times for 10 seconds each.
    3. Repeat in xylene, incubating sections two times for 10 seconds each.
  18. Mount coverslips.

posted June 2005

revised February 2008

protocol id: 295

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Flow Cytometry General Protocol

If using whole blood, please follow the Flow Cytometry Whole Blood Protocol.

A. Solutions and Reagents

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

  1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix.
  2. 16% Formaldehyde (methanol free).
  3. 100% methanol.
  4. Incubation Buffer: Dissolve 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.
  5. Recommended Anti-Mouse secondary antibodies::

B. Fixation

  1. Collect cells by centrifugation and aspirate supernatant.
  2. Resuspend cells in 0.5–1 ml 1X PBS. Add formaldehyde to obtain a final concentration of 4%.
  3. Fix for 10 min at 37°C.
  4. Chill tubes on ice for 1 min.
  5. For extracellular staining with antibodies that do not require permeabilization, proceed to immunostaining (Section D) or store cells in PBS with 0.1% sodium azide at 4°C; for intracellular staining, proceed to permeabilization (Section C).

C. Permeabilization

NOTE: This step is critical for many CST antibodies.

  1. Permeabilize cells by adding ice-cold 100% methanol slowly to pre-chilled cells, while gently vortexing, to a final concentration of 90% methanol. Alternatively, remove fix prior to permeabilization by centrifugation and resuspend in 90% methanol as described above.
  2. Incubate 30 min on ice.
  3. Proceed with immunostaining (Section D) or store cells at -20°C in 90% methanol.

D. Immunostaining

NOTE: Account for isotype matched controls for monoclonal antibodies or species matched IgG for polyclonal antibodies. Count cells using a hemocytometer or alternative method.

  1. Aliquot 0.5–1 x 106 cells into each assay tube (by volume).
  2. Add 2–3 ml incubation buffer to each tube and wash by centrifugation. Repeat.
  3. Resuspend cells in 100 μl of diluted primary antibody (prepared in incubation buffer at the recommended dilution).
  4. Incubate for 1 hr at room temperature.
  5. Wash by centrifugation in 2–3 ml incubation buffer.
  6. Resuspend cells in fluorochrome-conjugated secondary antibody, diluted in incubation buffer at the recommended dilution.
  7. Incubate for 30 min at room temperature.
  8. Wash by centrifugation in 2–3 ml incubation buffer.
  9. Resuspend cells in 0.5 ml PBS and analyze on flow cytometer; alternatively, for DNA staining, proceed to optional DNA stain (Section E).

E. Optional DNA Dye

  1. Resuspend cells in 0.5 ml of DNA dye (e.g. Propidium Iodide (PI)/RNase Staining Solution #4087).
  2. Incubate for at least 30 min at room temperature.
  3. Analyze cells in DNA staining solution on flow cytometer.

posted June 2005

Flow Cytometry Whole Blood Protocol

If using cell lines, please follow the Flow Cytometry General Protocol.

A. Solutions and Reagents

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

  1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix.
  2. 16% Formaldehyde (methanol free).
  3. Triton™ X-100: To prepare 50 ml of 0.1% Triton™ X-100 add 50 μl Triton™ X-100 to 50 ml 1 X PBS and mix well.
  4. 50% methanol.
  5. Incubation Buffer: Dissolve 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.
  6. Secondary Antibodies:

B. Preparation of Whole Blood (fixation, lysis, and permeabilization) for Immunostaining

  1. Aliquot 100 μl fresh whole blood per assay tube.
  2. OPTIONAL: Place tubes in rack in 37°C water bath for short-term treatments with ligands, inhibitors, drugs, etc.
  3. Add 65 μl of 10% formaldehyde to each tube.
  4. Vortex briefly and let stand for 15 min at room temperature.
  5. Add 1 ml of 0.1% Triton™ X-100 to each tube.
  6. Vortex and let stand for 30 min at room temperature.
  7. Add 1 ml incubation buffer.
  8. Pellet cells by centrifugation and aspirate supernatant.
  9. Repeat steps 7 and 8.
  10. Resuspend cells in ice-cold 50% methanol in PBS (store methanol solution at -20°C until use).
  11. Incubate at least 10 min on ice.
  12. Proceed with staining or store cells at -20°C in 50% methanol.

C. Staining Using Unlabeled Primary and Conjugated Secondary Antibodies

NOTE: Account for isotype-matched controls for monoclonal antibodies or species matched IgG for polyclonal antibodies.

  1. Add 2–3 ml incubation buffer to each tube and rinse by centrifugation. Repeat.
  2. Add primary antibodies diluted as recommended on datasheet or product webpage in incubation buffer.
  3. Incubate for 30–60 min at room temperature.
  4. Wash by centrifugation in 2–3 ml incubation buffer.
  5. Resuspend cells in fluorochrome-conjugated secondary antibody diluted in incubation buffer according to the manufacturer’s recommendations.
  6. Incubate for 30 min at room temperature.
  7. Wash by centrifugation in 2–3 ml incubation buffer.
  8. Resuspend cells in 0.5 ml PBS and analyze on flow cytometer.

Reference: Chow S, Hedley D, Grom P, Magari R, Jacobberger JW, Shankey TV (2005) Whole blood fixation and permeabilization protocol with red blood cell lysis for flow cytometry of intracellular phosphorylated epitopes in leukocyte subpopulations. Cytometry A 67(1), 4–17.

posted November 2008

revised September 2013

protocol id: 406

Product Usage Information

Application Dilutions
Immunohistochemistry 1:50
Flow Cytometry 1:400

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

CD34 (ICO115) Mouse mAb detects endogenous levels of total CD34 protein.


Species Reactivity: Human

Source / Purification

Monoclonal antibody is produced by immunizing BALB/c mice with blast cells of a chronic myeloid leukemia patient.

CD34 is a type I transmembrane glycophosphoprotein expressed by hematopoietic stem/progenitor cells, vascular endothelium and some fibroblasts (1). CD34 expression has been the hallmark used to identify hematopoietic stem cells for many years. CD34+ hematopoietic stem cells expand and differentiate into all the lymphohematopoietic lineages upon cytokine or growth factor stimulation and lose CD34 expression upon differentiation. However, recent studies performed in various laboratories conflict with that convention (2). The extracellular domain of CD34 is homologous to CD43, a protein involved in cell-cell adhesion, and CD34 has been shown to function as a negative regulator of cell adhesion (3). CD34 associates with CrkL but not CrkII, is a substrate for PKC, and activation of PKC is coupled with surface expression of CD34 (1,4).


1.  Felschow, D. M. et al. (2001) Blood 97, 3768-3775.

2.  Sato, T. et al. (1999) Blood 94, 2548-2554.

3.  Drew, E. et al. (2005) Immunity 22, 43-57.

4.  Fackler, M. J. et al. (1992) J. Biol. Chem. 267, 17540-17546.


Entrez-Gene Id 947
Swiss-Prot Acc. P28906


For Research Use Only. Not For Use In Diagnostic Procedures.
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