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Amyloid Precursor Protein Antibody Staining Protocol for Immunohistochemistry
Description: Different isoforms of amyloid precursor protein (APP) exist as a result of alternative splicing of a 19-exon gene. APP695, APP751 and APP770 are the predominant APP isoforms. In brain neuronal tissue APP695 is predominantly expressed while APP751 and APP770 are found in other tissues. APP, a transmembrane glycoprotein, plays a role in intracellular signalling by regulating cell-cell or cell-substrate interactions. It is involved in synaptogenesis. Normal synthesis of APP by nerve cells involves cleavage and release from the cell surface, but abnormal cleavage of APP results in a fragment called beta amyloid peptide that aggregates and forms plaques. These plaques are associated with Alzheimer’s Disease.
Primary Antibody
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Name: Amyloid Precursor Protein Antibody |
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Clone: 3G12, Mouse Anti-Human |
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Supplier: Novocastra Labs |
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Catalog Number: NCL-APP-228 |
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Dilution: 1:25 - 1:50 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: Neurofibrillary tangles and senile plaques |
| Images: Search image |
Additional Information:
| Tissue Type: Human brain, Alzheimer’s disease |
| Fixation: Formalin-fixed paraffin sections |
| Positive Control: Human brain, Alzheimer’s disease |
| 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: