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Cytokeratin AE1/AE3 Antibody Staining Protocol for Immunohistochemistry
Description: Keratins are a group of water-insoluble proteins that form monofilaments, a class of intermediate filament. These filaments form part of the cytoskeletal complex in epidermis and in most other epithelial tissues. Nineteen human epithelial keratins are resolved with two-dimensional gels electrophoresis (Moll et al., 1982). These can be divided into acid (pI <5.7) and basic (pI >6.0) subfamilies. The acidic keratins have molecular weights of 56.5, 55, 51, 50, 50’, 48, 46, 45, and 40 kD. The basic keratins have molecular weights of 65-67, 64, 59, 58, 56, and 52 kD.Members of the acidic and basic subfamilies are found together in pairs. The composition of keratin pairs varies with the epithelial cell type, stage of differentiation, cellular growth environment, and disease state.
Specificity: The
stringent, but broad, specificity of pooled AE1/AE3 antibody has
made this preparation very useful as a general stain for normal and
neoplastic cells of epithelial origin. Anti-Keratin AE1 recognizes
the 56.5, 50, 50’, 48, and 40 kD keratins of the acidic subfamily.
Anti-keratin AE3 recognizes all members of the basic subfamily,
65-67, 64.9, 58, 56, and 52kDa.
This antibody reacts with keratinized (56.5/65-67) and corneal
(55/64) epidermis, stratified squamous epithelium of internal organs
(51/59), stratified epithelia (50/58), hyperproliferative
keratinocytes (48/56), and simple epithelia (45/52 and 46/54). The
40kD keratin is present in most spithelia except adult epidermis.
Primary Antibody
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Name: Cytokeratin AE1/AE3 (Pan Cytokeratins) Antibody |
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Clone: AE1/AE3, Mouse anti-Human |
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Supplier: Chemicon |
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Catalog Number: MAB3412 |
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Dilution: 1:100 - 1:200 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: Incubator |
| Buffer/pH value: IHC-TekTM Proteinase K Solution (Cat# IW-1101) |
| Heat/Cool Temperature: 37 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: Skin |
| Fixation: Formalin fixed paraffin sections, or acetone fixed frozen sections |
| Positive Control: Skin |
| 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: