Cdk4 Antibody Staining Protocol for Immunohistochemistry


Description: Cell cycle progression is controlled in part by a family of cyclin proteins and cyclin dependent kinases (Cdks). Cdk proteins work in concert with the cyclins to phosphorylate key substrates involved in each phase of cell cycle progression. Another family of proteins, Cdk inhibitors, also plays a role in regulating the cell cycle by binding to cyclin-Cdk complexes and modulating their activity. Several Cdk proteins have been identified, including Cdk2–Cdk8, PCTAIRE-1–3, PITALRE and PITSLRE. Cdk4, in complex with D-type cyclins, is thought to regulate cell growth during the G1 phase of the cell cycle. This association with a D-type cyclin upregulates Cdk4 activity, whereas binding to the Cdk inhibitor p16 downregulates Cdk4 activity. Activation of the Cdk4-cyclin complexes requires phosphorylation on a single threonyl residue of Cdk4, catalyzed by a Cdk-activating protein (CAK).


Primary Antibody

Name: Cdk4 Antibody

Clone: Rabbit anti-Cdk4

Supplier: Santa Cruz Biotechnology

Catalog Number: sc-260

Dilution: 1:5000 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: Nuclear
Images: Search image

Additional Information:
Tissue Type: Skin, NIH/3T3 cells
Fixation: Formalin fixed paraffin sections, or methanol fixed frozen sections
Positive Control: Skin, NIH/3T3 cells
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