Bcl-2 Oncoprotein Antibody Staining Protocol for Immunohistochemistry


Description: In 90% of follicular lymphomas a translocation occurs with juxtaposes the bcl-2 gene at 18q21, to an immunoglobulin gene, with subsequent deregulation of protein synthesis and cell proliferation. The bcl-2 product is considered to act as an inhibitor of apoptosis. For this reason, bcl-2 expression is inhibited in germinal center where apoptosis forms part of the B cell production pathway. This observation has tumed out to have clinical implications. Distribution of follicular hyperplasia form follicular lymphoma is a common problem in histopathology. Reactive follicles show no staining for bcl-2, whereas the cells in neoplastic follicules exhibit membrane and/or cytoplasmic staining.


Primary Antibody

Name: Bcl-2 Oncoprotein Antibody

Clone: 124, Mouse Anti-Human

Supplier: Dako

Catalog Number: M0887

Dilution: 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: 60min/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: 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