Histone H2AX Antibody Staining Protocol for Immunohistochemistry


Description: Histone H2AX is a 14 kDa ubiquitous member of the H2A histone family that contains an evolutionary conserved SQ motif at the C-terminus in eukaryotes. Serine 139 within this motif becomes rapidly phosphorylated to yield a form known as γ-H2AX in response to double-strand DNA damage and apoptosis. Phosphorylation reaches half its maximum between 1-3 minutes after DNA damage occurs, and hundreds to several thousand molecules of γ-H2AX are present per double-strand break. This antibody is unique in only detecting double strand DNA breaks.


Primary Antibody

Name: Histone H2AX Antibody

Clone: Rabbit polyclonal

Supplier: Trevigen

Catalog Number: 4411-PC-100

Dilution: 1:100 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, skin

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.