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-Tek^TM 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-Tek^TM Epitope Retrieval Steamer Set (Cat# IW-1102)

Buffer/pH value: IHC-Tek^TM 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:

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

Notes:

Not tested on frozen sections.

References: