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

Name: phospho-Histone H3 Antibody

Clone: Rabbit anti-Human

Supplier: Upstate

Catalog Number: 06-570

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.

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

Reagent: Mayer's Hematoxylin
Staining Time: 30 seconds

Staining Pattern: Nuclear
Images: Search image

Additional Information:

Tissue Type: Tonsil, skin

Fixation: Formalin fixed paraffin sections

Positive Control: Tonsil, cervix, skin, Alzheimer's brain, colonic adenocarcinoma, carcinoma.

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


Not tested on frozen sections.



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.