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

Name: Amyloid Precursor Protein Antibody

Clone: 3G12, Mouse Anti-Human

Supplier: Novocastra Labs

Catalog Number: NCL-APP-228

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.

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: 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