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Cdc2 Antibody Staining Protocol for Immunohistochemistry
Description: Entry of all eukaryotic cells into M-phase of the cell cycle is regulated by activation of cdc2 kinase. Activation of cdc2 is controlled at several steps including cyclin binding and phosphorylation of threonine 161. However, the critical regulatory step in activating cdc2 during progression into mitosis appears to be dephosphorylation of tyrosine 15 and threonine 14. Phosphorylation at Tyr15 and inhibition of cdc2 is carried out by Wee1 and Myt1 protein kinases while Tyr15 dephosphorylation and activation of cdc2 is carried out by the cdc25 phosphatase. This antibody detects total levels of endogenous cdc2, but does not cross react with other cdk's.
Primary Antibody
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Name: Cdc2 Antibody |
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Clone: POH-1, Mouse anti-Cdc2 |
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Supplier: Cell Signaling Technology |
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Catalog Number: 9116 |
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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. |
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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: Small intestine, breast carcinoma, tonsil, lymphoma. |
| Fixation: Formalin fixed paraffin embedded sections |
| Positive Control: Small intestine, breast carcinoma, tonsil, lymphoma |
| 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