GFAP Antibody Staining Protocol for Immunohistochemistry


Description: Glial fibrillary acidic protein (GFAP) is an intermediate filament protein of 52kD found in glial cells such as astrocytes and ependymal cells. In the peripheral nervous system, GFAP has been demonstrated in Schwann cells, enteric glial cells and satellite cells of human sensory ganglia. In neoplastic tissues, this antibody is useful for the identification of astrocytomas and ependymomas.  


Primary Antibody

Name: GFAP Antibody

Clone: GA5, Mouse anti-Human

Supplier: Novocastra

Catalog Number: NCL-GFAP-GA5

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 minutes/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 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 (glial cells such as astrocytes and ependymal cells)
Images: Search image

Additional Information:
Tissue Type: Brain (cerebellum, striatum)
Fixation: Formalin fixed paraffin sections, or acetone fixed frozen sections
Positive Control: Brain (cerebellum, striatum)
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