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Involucrin Antibody Staining Protocol for Immunohistochemistry
Description: Involucrin is a 68 kDa precursor protein of the keratinocyte cornified envelope, which is formed beneath the inner surface of the cell membrane during terminal differentiation. Involucrin first appears in the cell cytosol but ultimately becomes cross-linked to membrane proteins by transglutaminase. During keratinocyte terminal differentiation glutamine residues of involucrin become covalently cross-linked to other envelope precursors via covalent epsilon-(gamma-glutamyl) lysine bonds. Moreover, its large size allows involucrin to cross-link molecules that are separated by substantial distances in the cornified envelope. These properties allow a single involucrin molecule to form multiple cross-links, in multiple spatial planes, with other envelope precursors. Involucrin is specifically expressed in Chinese hamster ovarian cells (fibroblasts), PtK2 rat kangaroo kidney cells (simple epithelial), and rat epidermal keratinocytes (stratifying squamous epithelial).
Primary Antibody
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Name: Involucrin (N-17) |
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Clone: Polyclonal Goat ant-Human |
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Supplier: Santa Cruz Biotechnology |
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Catalog Number: sc-15223 |
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Dilution: 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 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 ºC - 100 ºC/room temperature |
| Heat/Cool Time: 20 minutes/20 minuters |
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:
| Species Reactivity: Human |
| Fixation: Formalin fixed paraffin sections. |
| Positive Control: Human skin (epidermis) |
| 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 |
References: