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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
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Name: Melan-A Antibody |
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Clone: A103, Mouse anti-Human |
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Supplier: Dako |
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Catalog Number: M7196 |
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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. |
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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 |
Counterstain
| Reagent: Mayer's Hematoxylin |
| Staining Time: 30 seconds |
Results:
| 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 |
Note:
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.
References: