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REACTIVITY SENSITIVITY MW (kDa) Isotype
H Endogenous 70-80 Mouse IgG2a
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Immunohistochemistry (Paraffin)

Immunohistochemical analysis of human colon tissue using Tyrosinase (T311) Mouse mAb (IHC Specific). Note the lack of staining in the tyrosinase negative tissue.

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Immunohistochemistry (Paraffin)

Immunohistochemical analysis of human melanoma tissue using Tyrosinase (T311) Mouse mAb (IHC Specific).

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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:
    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. Antibody Diluent: SignalStain® Antibody Diluent (#8112)
  7. Antigen Unmasking: TE: 10 mM Tris/1 mM EDTA, pH 9.0: To prepare 1L add 1.21 g Trizma® base (C4H11NO3) and 0.372 g EDTA (C10H14N2O8Na2·2H2O) to 950 ml dH2O. Adjust pH to 9.0, then adjust final volume to 1000 ml with dH2O.
  8. 3% Hydrogen Peroxide: To prepare, 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 Reagent (HRP, Mouse) (#8125).
  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 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

  1. For TE: Heat slides in a microwave submersed in TE pH 9.0 until boiling is initiated; follow with 18 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 minutes each.
  2. Incubate sections in 3% hydrogen peroxide for 10 minutes.
  3. Wash sections in dH2O twice for 5 minutes each.
  4. Wash section in wash buffer for 5 minutes.
  5. Block each section with 100-400 µl of preferred blocking solution for 1 hour at room temperature.
  6. Remove blocking solution and add 100-400 µl primary antibody diluted in SignalStain® Antibody Diluent (#8112) to each section. Incubate 30 minutes at room temperature.
  7. Equilibrate SignalStain® Boost IHC Detection Reagent (HRP, Mouse) (#8125) reagent to room temperature.
  8. Remove antibody solution and wash sections in wash buffer three times for 5 minutes each.
  9. Cover section with 1-3 drops SignalStain® Boost Detection Reagent (HRP, Mouse #8125) as needed. Incubate in a humidified chamber for 30 min at room temperature.
  10. Wash sections three times with wash buffer for 5 minutes 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 minutes each.
  16. 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.
  17. Mount sections with coverslips and mounting medium (#14177).

posted November 2010

revised June 2016

Protocol Id: 70

Product Usage Information

Application Dilutions
Immunohistochemistry (Paraffin) 1:200

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

Tyrosinase (T311) Mouse mAb detects endogenous levels of total tyrosinase protein.


Species Reactivity: Human

Source / Purification

Tyrosinase (T311) Mouse mAb is produced by immunizing animals with a purified recombinant tyrosinase.

Tyrosinase, found in the membrane of melanosomes (1), is a key enzyme in the biosynthesis of melanin pigments (1,2). It is a melanocyte differentiation antigen and is expressed in normal melanocytes and malignant melanomas (1,3). Tyrosinase is implicated to be an antigen target for melanoma vaccines (1).


1.  Chen, Y.T. et al. (1995) Proc Natl Acad Sci USA 92, 8125-9.

2.  Kwon, B.S. (1993) J Invest Dermatol 100, 134S-140S.

3.  Kwon, B.S. et al. (1987) Proc Natl Acad Sci USA 84, 7473-7.


Entrez-Gene Id 7299
Swiss-Prot Acc. P14679


For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.

9319
Tyrosinase (T311) Mouse mAb (IHC Specific)