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Estrogen Receptor (ER) Antibody Staining Protocol for Immunohistochemistry
Description: Steroid receptors exhibit a high affinity and specificity for their ligands. The human estrogen receptor (ER) is a dimeric protein of 65 kDa located primarily on the membrane of cell nuclei and belongs to a class of trans-acting proteins which stimulate transcription by binding to specific DNA elements, also known as hormone response elements. Through binding estrogen, the ER is induced to stimulate gene transcription, hence is also known as an inducible enhancer factor. NCL-ER-6F11, effective on formalin-fixed, paraffin-embedded material, allows the determination of ER in routinely processed and archived material.
Primary Antibody
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Name: Estrogen Receptor (ER) Antibody |
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Clone: 6F11, Mouse anti-Human |
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Supplier: Novocastra |
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Catalog Number: NCL-ER-6F11 |
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Dilution: 1:100 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: Nuclear |
| Images: Search image |
Additional Information:
| Tissue Type: Breast Mammary gland, Cervix uteri squamous epithelium, Prostate fibromuscular stroma, Uterus Endometrium. |
| Fixation: Formalin fixed paraffin sections, or acetone fixed frozen sections. |
| Positive Control: Breast Mammary gland, Cervix uteri squamous epithelium, Prostate fibromuscular stroma, Uterus Endometrium. |
| 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:
No need to perform antigen retrieval on frozen sections |
References:
1. Charafe-Jauffret E, et al (2004) Immunophenotypic analysis of inflammatory breast cancers: identification of an inflammatory signature. J Pathology. 202:265-273.