4-Hydroxy-2-nonenal (HNE) Antibody Staining Protocol for Immunohistochemistry

 

Description: HNE is a major product of endogenous lipid peroxidation. The w-6-family of polyunsaturated fatty acides may produce HNE as a result of free radical attack. HNE is a highly reactive compound and it can react with several functional groups on biological material, particularly sulfydryl groups to form thioester adduct and then hemiacetals. HNE may also react with histidine and lysine residues of proteins to form stable Michael addition-type of adducts. HNE-modified proteins may display an altered biological functions. Antibodies to HNE will help to visualize the HNE-adducts.

 

Primary Antibody

Name: HNE Antibody

Clone: Rabbit anti-Rat

Supplier: Alpha Diagnostic

Catalog Number: HNE11-S

Dilution: 1:500 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

Counterstain

Reagent: Mayer's Hematoxylin

Staining Time: 30 seconds

Results:

Staining Pattern: Cytoplasmic

Images: Search image

Additional Information:

Tissue Type: Liver

Fixation: Formalin fixed paraffin sections

Positive Control: Liver

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: