Alpha-1-Antitrypsin Antibody Staining Protocol for Immunohistochemistry

Alpha 1 Antitrypsin Antibody Staining Protocol for Immunohistochemistry

Description: Alpha-1-antitrypsin is synthesised in the liver and is present in serum and tissue fluids where it acts as an inhibitor of proteases, particularly elastase. Its main function appears to be the neutralisation of elastase released by neutrophils during an inflammatory response. As a result, in alpha-1-antitypsin deficiency, any stimulus which activates neutrophils in the lung may result in uninhibited elastase-induced tissue destruction.

Primary Antibody

Name: alpha-1-Antitrypsin Antibody

Clone: Rabbit Polyclonal

Supplier: Novocastra Labs

Catalog Number: NCL-A1Ap

Dilution: 1:250 - 1:500 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 min/room temperature

Antigen Retrieval

Device: Incubator

Buffer/pH value: IHC-Tek^TM Proteinase K Solution (Cat# IW-1101)

Heat/Cool Temperature: 37 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 staining of macrophages

Images: Search image

Additional Information:

Tissue Type: Colon, tonsil

Fixation: Formalin-fixed paraffin sections, and acetone fixed frozen sections

Positive Control: Colon, tonsil

Negative Control: Omit primary antibody, isotype control, 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:

There is no need to perform antigen retrieval on acetone fixed frozen sections

References:

1. Alpha-1-antitrypsin deficiency - Alpha-1 antitrypsin deficiency (A1AD) is an autosomal recessive disorder marked by abnormally low or no serum levels of alpha-1 antitrypsin in the blood. It is the most common genetic cause of liver disease in children and of emphysema in adults

2. Callea F, Fevery J, De Groote J, Desmet VJ. Detection of Pi Z phenotype individuals by alpha-1-antitrypsin immunohistochemistry in paraffin-embedded liver tissue specimens. J Hepatol 1986; 2:389-401. PubMed Abstract.

3. Carlson J, Eriksson S. Chronic 'cryptogenic' liver disease and malignant hepatoma in intermediate alpha-1-antitrypsin deficiency identified by a Pi Z-specific monoclonal antibody. Scand J Gastroenterol. 1985; 20:835-842. PubMed Abstract.