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

Name: Cytokeratin AE1/AE3 (Pan Cytokeratins) Antibody

Clone: AE1/AE3, Mouse anti-Human

Supplier: Chemicon

Catalog Number: MAB3412

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.

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

Reagent: Mayer's Hematoxylin
Staining Time: 30 seconds

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


No need to perform antigen retrieval on frozen sections