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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
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Name: Neurofilament Antibody |
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Clone: 2F11, Mouse anti-Human |
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Supplier: Dako |
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Catalog Number: M0762 |
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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. |
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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: 95-100 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 |
Counterstain
| Reagent: Gill's Hematoxylin or Mayer's Hematoxylin |
| Staining Time: 30 seconds |
Results:
| 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 |
References: