Neurofilament Antibody Staining Protocol for Immunohistochemistry


Description: Neurofilaments are main structural elements of neurons and their processes and are comprised of a triad of distinct proteins with molecular weights of approximately 70, 160 and 200 kDa. Anti-NF, 2F11 reacts with the phosphorylated forms of the 70 and 200 kDa components of the three major polypeptide units. The antibody shows interspecies cross-reactivity, reacting with neurofilament from other species, e.g. mouse, rat and rabbit, but does not recognize other intermediate filament proteins.


Primary Antibody

Name: Neurofilament Antibody

Clone: 2F11, Mouse anti-Human

Supplier: Dako

Catalog Number: M0762

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: 95-100 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
Images: Search image

Additional Information:
Species Reactivity: Human, rat, mouse, rabbit
Fixation: Formalin fixed paraffin sections, acetone fixed frozen sections, or cell smears (no antigen retrieval needed for frozen sections or cell smears)
Positive Control: Most of neurons and neuronal processes in central and peripheral nerves
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