Actin (alpha-Sarcomeric) Antibody Staining Protocol for Immunohistochemistry

 

Description: This antibody is specific for the single isoform of alpha-cardiac muscle actins. It reacts with a-skeletal and a-cardiac muscle actins. It does not react with smooth muscle tissue.

 

Primary Antibody

Name: Actin (alpha-Sarcomeric)

Clone: 5C5, Mouse

Supplier: Sigma-Aldrich

Catalog Number: A2172

Dilution: 1:1000 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 min/room temperature

Antigen Retrieval

Device: Antigen retrieval is not needed.

Buffer/pH value: Citrate buffer reduce staining intensity, NOT enhance!

Heat/Cool Temperature: N/A

Heat/Cool Time: N/A

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: Rat cardiac muscle

Fixation: Formalin fixed paraffin secitons or acetone fixed frozen sections

Positive Control: Cardiac or skeletal muscle. Rat cardiac muscle works well.

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

 

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