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: bcl-2/100/D5, Mouse Anti-Human

Supplier: Novocastra Labs

Catalog Number: NCL-bcl-2

Dilution: 1:50

Incubation Time/Temp: 60min/room temperature

Antigen Retrieval

Device: Steamer

Buffer/pH value: Tris-EDTA/pH 9.0

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: Gill's Hematoxylin or 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, or acetone fixed frozen 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

Notes:

There is no need to perform antigen retrieval on acetone fixed frozen sections

 

References: