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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 colon carcinoma using CD63 (D4I1X) Rabbit mAb (IHC Specific).

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Immunohistochemistry (Paraffin)

lung:

Immunohistochemical analysis of paraffin-embedded human lung carcinoma using CD63 (D4I1X) Rabbit mAb (IHC Specific).

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Immunohistochemistry (Paraffin)

lymph:

Immunohistochemical analysis of paraffin-embedded human Non-Hodgkin's Lymphoma using CD63 (D4I1X) Rabbit mAb (IHC Specific).

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Immunohistochemistry (Paraffin)

Immunohistochemical analysis of paraffin-embedded human melanoma using CD63 (D4I1X) Rabbit mAb (IHC Specific).

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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

Product Usage Information

Application Dilutions
Immunohistochemistry (Paraffin) 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

CD63 (D4I1X) Rabbit mAb (IHC Specific) recognizes endogenous levels of total CD63 protein.


Species Reactivity: Human

Source / Purification

Monoclonal antibody is produced by immunizing animals with recombinant CD63 protein.

CD63 belongs to the tetraspanin family, which is characterized by four transmembrane domains, one short extracellular domain (ECL1), and one long extracellular domain (ECL2) (1-3). Tetraspanins interact with a variety of cell surface proteins and intracellular signaling molecules in specialized tetraspanin enriched microdomains (TEMs) where they mediate a range of processes including adhesion, motility, membrane organization, and signal transduction (3). CD63, like other tetraspanins, is enriched in exosomes (4). It is also a component of Weibel-Palade bodies found in endothelial cells (5). Research studies demonstrate several functions of CD63 in different cell types including roles in mast cell degranulation, VEGF signaling in endothelial cells, recruitment of leukocytes to endothelial cells, and endosomal sorting during melanogenesis (6-9).


1.  Oren, R. et al. (1990) Mol Cell Biol 10, 4007-15.

2.  Levy, S. et al. (1991) J Biol Chem 266, 14597-602.

3.  Hemler, M.E. (2005) Nat Rev Mol Cell Biol 6, 801-11.

4.  Escola, J.M. et al. (1998) J Biol Chem 273, 20121-7.

5.  Vischer, U.M. and Wagner, D.D. (1993) Blood 82, 1184-91.

6.  Kraft, S. et al. (2013) J Immunol 191, 2871-8.

7.  Tugues, S. et al. (2013) J Biol Chem 288, 19060-71.

8.  Doyle, E.L. et al. (2011) Blood 118, 4265-73.

9.  van Niel, G. et al. (2011) Dev Cell 21, 708-21.


Entrez-Gene Id 967
Swiss-Prot Acc. P08962


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.

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CD63 (D4I1X) Rabbit mAb (IHC Specific)