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

Name: Involucrin (N-17)

Clone: Polyclonal Goat ant-Human

Supplier: Santa Cruz Biotechnology

Catalog Number: sc-15223

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.

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

Reagent: Mayer's Hematoxylin
Staining Time: 30 seconds

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