Alpha Actinin Antibody Staining Protocol for Immunohistochemistry

 

Description: Alpha-actinin is a rod-like cytoskeletal protein belonging to the same family as spectrin, dystrophin and utrophin. In skeletal muscle, alpha-actinin is located in the Z band/disc and cross-links with F-actin in this region. Muscle tissues show the presence of abundant threadlike particles, known as nemaline bodies, in the myofibres. Electron microscopy studies have shown that the nemaline rods have a lattice structure similar to that of the Z discs and the rods are thought to be lateral polymers of the Z discs.

 

Primary Antibody

Name: alpha Actinin Antibody

Clone: RBC2/1B6, Mouse

Supplier: Novocastra Labs

Catalog Number: NCL-alpha-ACT

Dilution: 1:40 - 1:80 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: N/A

Buffer/pH value: N/A

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: Striations localized to the Z disc in longitudinal sections

Images: Search image

Additional Information:

Tissue Type: Striated muscle

Fixation: Acetone fixed frozen sections

Positive Control: Normal human striated muscle frozen in isopentane chilled in liquid nitrogen.

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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