Bcl-2 Oncoprotein Antibody Staining Protocol for Immunohistochemistry

Description: Bcl-2 is a member of a family of proteins that are involved in apoptosis. Bcl-2 is an integral inner mitochondrial membrane protein of 25kD and has a wide tissue distribution. It is considered to act as an inhibitor of apoptosis. For this reason, bcl-2 expression is inhibited in germinal centres where apoptosis forms part of the B cell production pathway.

Primary Antibody

Name: Bcl-2 Oncoprotein Antibody

Clone: 3.1, Mouse Anti-Human

Supplier: Novocastra Labs

Catalog Number: NCL-bcl-2-486

Dilution: 1:100 using IHC-Tek^TM Antibody Diluent (Cat# IW-1000 or IW-1001) to reduce background and unspecific staining and serum blocking step is NOT needed.

Incubation Time/Temp: 60min/room temperature

Antigen Retrieval

Device: IHC-Tek^TM Epitope Retrieval Steamer Set (Cat# IW-1102)

Buffer/pH value: IHC-Tek^TM 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: Membrane and cytoplasmic

Images: Search image

Additional Information:

Tissue Type: Tonsil

Fixation: Formalin-fixed paraffin sections.

Positive Control: Tonsil

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:

1. Backus HH, Van Groeningen CJ, Vos W, Dukers DF, Bloemena E, Wouters D, Pinedo HM, Peters GJ. (2002) Differential expression of cell cycle and apoptosis related proteins in colorectal mucosa, primary colon tumours, and liver metastases. J Clin Pathol. 55(3):206-11. PubMed Abstract.

2. Takes RP, Baatenburg de Jong RJ, Wijffels K, Schuuring E, Litvinov SV, Hermans J, van Krieken JH. (2001) Expression of genetic markers in lymph node metastases compared with their primary tumours in head and neck cancer. J Pathol. 194(3):298-302. PubMed Abstract.