Filaggrin Antibody Staining Protocol for Immunohistochemistry


Description: Profilaggrin is a large, insoluble, highly phosphorylated precursor protein containing several tandem copies of a 324 amino acid stretch. Mammalian profilaggrin is a major protein component of keratohyalin granules in the living cells of the epidermis. Keratohyalin granules contribute to the keratin content of dead cornified cells. During terminal differentiation of the epidermis, profilaggrin is proteolytically processed into active filaggrin molecules that promote aggregation and disulfide-bond formation of keratin intermediate filaments. Active filaggrin is present at a level of the epidermis where keratinocytes are in transition between the live nucleated granular layer and the anucleate cornified layer, suggesting that filaggrin aids in the terminal differentiation process by facilitating apoptotic machinery.


Primary Antibody

Name: Filaggrin (N-20)

Clone: Polyclonal Goat ant-Human

Supplier: Santa Cruz Biotechnology

Catalog Number: sc-25896

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.

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 of epidermal cells
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