 |
|
Cdc2 Antibody Staining Protocol for Immunohistochemistry
Description: During the eukaryotic cell cycle, activation of Cdc2 (Cdk1) kinase regulates the transition into M-phase. Cdc2 forms a complex with cyclin B, and activation of this complex is controlled by dephosphorylation of Cdc2 at tyrosine 15 and threonine 14. Phosphorylation at Tyr15 and is carried out by Wee1 and Myt1 protein kinases, while Tyr15 dephosphorylation is carried out by the cdc25 phosphatase.
Primary Antibody
|
Name: Cdc2 (Y15) (Non-phospho-specific) Antibody |
|
Clone: Rabbit monoclonal |
|
Supplier: Epitomics |
|
Catalog Number: 1161-1 |
|
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 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 |
Counterstain
| Reagent: Mayer's Hematoxylin |
| Staining Time: 30 seconds |
Results:
| Staining Pattern: Cytoplasmic/nuclear |
| Images: Search image |
Additional Information:
| Tissue Type: Tonsil, lymphoma, colorectal cancer |
| Fixation: Formalin fixed paraffin sections |
| Positive Control: Tonsil, lymphoma, colorectal cancer |
| 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. Hui D, Reiman T, Hanson J, Linford R, Wong W, Belch A, Lai R (2005) Immunohistochemical detection of cdc2 is useful in predicting survival in patients with mantle cell lymphoma. Mod Pathol. 2005 Sep;18(9):1223-31. PubMed Abstract
2. Yamamoto H, et al (1998) Cdk2/cdc2 expression in colon carcinogenesis and effects of cdk2/cdc2 inhibitor in colon cancer cells. Int J Oncol. 13(2):233-9. PubMed Abstract
3. Winters ZE, et al (2001) Subcellular localisation of cyclin B, Cdc2 and p21(WAF1/CIP1) in breast cancer. association with prognosis. Eur J Cancer. 37(18):2405-12. PubMed Abstract
4. Tsujioka Y, et al (1999) Localization and expression of cdc2 and cdk4 in Alzheimer brain tissue. Dement Geriatr Cogn Disord. 10(3):192-8. PubMed Abstract
5. Yamamoto H, et al (1995) Coexpression of cdk2/cdc2 and retinoblastoma gene products in colorectal cancer. Br J Cancer. 71(6):1231-6. PubMed Abstract
6. De Souza CP, Ellem KA, Gabrielli BG (2000) Centrosomal and cytoplasmic Cdc2/cyclin B1 activation precedes nuclear mitotic events. Exp Cell Res. 25;257(1):11-21. PubMed Abstract
7. Yasuda H, Kamijo M, Honda R, Nagahara M, Ohba Y (1990) The difference in murine CDC2 kinase activity between cytoplasmic and nuclear fractions during the cell cycle. Biochem Biophys Res Commun. 172(2):371-6. PubMed Abstract