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Human Chorionic Gonadotropin (hCG) Antibody Staining Protocol for Immunohistochemistry
Description: Human chorionic gonadotropin (hCG) is a glycoprotein hormone synthesized in syncytiotrophoblastic cells of placenta and in certain trophoblastic tumors. Maintenance of the foetus in the first trimester of pregnancy requires the production of hCG, which binds to the corpus luteum of the ovary which is stimulated to produce progesterone which in turn maintains the secretory endometrium. hCG is composed of two subunits, alpha and beta. The alpha subunit of hCG is identical to the subunit of luteinising hormone, thyroid stimulating hormone and follicle stimulating hormone. The common alpha chain and the hormone-specific beta chains have molecular weights of 14kD and 17kD, respectively. The hCG beta-subunit is unique in the family of beta-containing glycoprotein hormones in that it contains an extension of 29 amino acids at its COOH end. It is believed that the C-terminal region of the CG-beta subunit plays a role in the intracellular behaviour of the heterodimer. This antibody labels the cytoplasm of syncytiotrophoblastic cells and their tumors, as well as germ cell tumors of the ovaries, testes and extragonadal sites.
Primary Antibody
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Name: hCG Antibody |
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Clone: Rabbit anti-Human |
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Supplier: Dako |
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Catalog Number: A0231 |
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Dilution: 1:20,000 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 minutes/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 ºC - 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: Placenta, ovaries, testis. |
| Fixation: Formalin fixed paraffin sections, or acetone fixed frozen sections |
| Positive Control: Placenta, ovaries, testis. |
| Negative Control: Omit primary antibody, isotype control or 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 |
Notes:
1. No need to perform antigen retrieval on frozen sections 2. hCG (Novocastra, Cat# NCL-HCG-p) also works well at 1:500 dilution with Proteinase K or Trypsin pretreatment method. |
References: