Cyclin D1 Antibody Staining Protocol for Immunohistochemistry

 

Description: Cyclin D1 or PRAD-1 or bcl-1 is one of the key cell cycle regulators, and functions in association with cdk4 and / or cdk6 by phosphorylating the Rb protein. It is a putative proto-oncogene overexpressed in a wide variety of human neoplasms including mantle cell lymphomas (MCL).

 

Primary Antibody

Name: Cyclin D1 Antibody

Clone: SP4, Rabbit monoclonal

Supplier: Lab Vision

Catalog Number: RM-9104-S1

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: 60 minutes/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 ºC - 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: Cytoplasmic
Images: Search image

Additional Information:
Tissue Type: MAD109 cells, Breast carcinomas or mantle cell lymphoma
Fixation: Formalin fixed paraffin sections
Positive Control: MAD109 cells, Breast carcinomas or mantle cell lymphoma
Negative Control: Omit primary antibody, isotype control or 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:

Not tested on frozen sections

 

References: