Melan-A Antibody Staining Protocol for Immunohistochemistry


Description: Melan-A is expressed in the majority of human melanomas as well as in melanocytes. The Melan-A gene, also called MART-1, encodes a melanoma specific antigen recognized by autologous cytotoxic lymphocytes.  This antibody reacts with many tumor cells: Benign and malignant adrenocortical tumors, Leydig tumors of the testis, and Sertoli-Leydig ovarian tumors.


Primary Antibody

Name: Melan-A Antibody

Clone: A103, Mouse anti-Human

Supplier: Dako

Catalog Number: M7196

Dilution: 1:30 using IHC-TekTM Antibody Diluent (Cat# IW-1000 or IW-1001) to reduce background and unspecific staining and serum blocking step is NOT needed.

Incubation Time/Temp: 60 min/room temperature

Antigen Retrieval
Device: IHC-TekTM Epitope Retrieval Steamer Set (Cat# IW-1102)
Buffer/pH value: IHC-TekTM Epitope Retrieval Solution (Cat# IW-1100)
Heat/Cool Temperature: 95-100 ºC/room temperature
Heat/Cool Time: 20 minutes/20 minutes

Detection Methods
Standard Method: ABC Method or LSAB Method
Enhanced Method: Polymeric Methods

Chromogen Substrate
Reagent: DAB
Incubation Time/Temperature: 1-3 minutes/room temperature

Reagent: Mayer's Hematoxylin
Staining Time: 30 seconds

Staining Pattern: Cytoplasmic
Images: Search image

Additional Information:
Tissue Type: Testis tumors, ovarian tumors, CaC1 melanoma cells and Melanomas.
Fixation: Formalin fixed paraffin sections, acetone fixed frozen sections, or cytospin preparations.
Positive Control: Testis tumors, ovarian tumors, CaC1 melanoma cells and Melanomas.
Negative Control: Omit primary antibody, isotype control, absorption control
Blocking: 2-5% normal serum to reduce unspecific background staining; 0.5-3% H2O2 to block endogenous peroxidase activity; avidin/biotin to block endogenous biotin activity if necessary


Melan-A/MART-1 (Clone M2-7C10 + M2-9E3, Labvision, Cat# MS-716-P1) also works well at 1:100 dilution on paraffin sections with Citrate Buffer Method.