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phospho-Histone H3 Antibody Staining Protocol for Immunohistochemistry
Description: Histone H3 (Mitosis Marker) is specific for phosphorylated histone H3, Mr 17kDa and has been reported to detect mitotic cells in C. elegans and Drosophila embryos by immunohistochemistry.
Primary Antibody
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Name: phospho-Histone H3 Antibody |
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Clone: Rabbit anti-Human |
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Supplier: Upstate |
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Catalog Number: 06-570 |
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Dilution: 1:200 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 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: Gill's Hematoxylin or Mayer's Hematoxylin |
| Staining Time: 30 seconds |
Results:
| Staining Pattern: Nuclear |
| Images: Search image |
Additional Information:
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Tissue Type: Tonsil, skin |
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Fixation: Formalin fixed paraffin sections |
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Positive Control: Tonsil, cervix, skin, Alzheimer's brain, colonic adenocarcinoma, carcinoma. |
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Negative Control: Omit primary antibody, isotype control or absorption control |
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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:
1. Ogawa O, et al (2003) Ectopic localization of phosphorylated histone H3 in Alzheimer's diseases: a mitotic catastrophe? Acta Neuropathol 105:524-528.
2. Scott IS, et al (2003) A novel immunohistochemical method to estimate cell-cycle phase distribution in archival tissue: implications for the prediction of outcome in colorectal cancer. J Pathology 201:187-197.