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Alpha Synuclein Antibody Staining Protocol for Immunohistochemistry
Description: Alpha-synuclein is a protein of 140 amino acids and a member of the synuclein family. It shares sixty one per cent of its sequence with beta-synuclein and is highly conserved
between vertebrate species. It does not possess a signal sequence suggesting that it is an
intracellular protein. All synucleins have the unusual organisation based around an eleven residue repeating motif and an alpha-helical secondary structure resembling those found in the lipid-binding domain of exchangeable apolipoproteins including Apo E. This homology suggests a direct interaction of alpha-synuclein with membranes consistent with its affinity for synaptosomes. The function of alpha-synuclein may be to carry a target protein to the inner membrane of nerve terminals or to the outer surface of synaptic vesicles.
Primary Antibody
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Name: alpha-Synuclein Antibody |
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Clone: KM51, Mouse |
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Supplier: Novocastra Labs |
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Catalog Number: NCL-ASYN |
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Dilution: 1:20 - 1:40 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: Mayer's Hematoxylin |
| Staining Time: 30 seconds |
Results:
| Staining Pattern: Lewy body and neuropil staining |
| Images: Search image |
Additional Information:
| Tissue Type: Posterior cingulated gyrus (Lewy body dementia). |
| Fixation: Formalin-fixed paraffin sections |
| Positive Control: Posterior cingulated gyrus (Lewy body dementia). |
| 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: