Connexin 43 Antibody Staining Protocol for Immunohistochemistry


Description: Synthetic peptide conjugated with glutaraldehyde to KLH. The peptide corresponds to the C-terminal segment of the cytoplasmic domain (amino acid residues 363-382 with N-terminally added lysine) of human/rat connexin-43.


Primary Antibody

Name: Connexin 43 Antibody

Clone: Rabbit polyclonal

Supplier: Sigma-Aldrich

Catalog Number: C6219

Dilution: 1:1500 - 1:2000 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: Antigen retrieval is not neccessary for this antibody. IHC-TekTM Epitope Retrieval Solution (Cat# IW-1100) enhances staining intensity.
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

Reagent: Mayer's Hematoxylin
Staining Time: 30 seconds

Staining Pattern: Cytoplasmic/membrane (cardiac muscle z line)
Images: Search image

Additional Information:
Tissue Type: Cardiac or skeletal muscle. Rat cardiac muscle.
Fixation: Formalin fixed paraffin sections, or acetone fixed frozen sections.
Positive Control: Cardiac or skeletal muscle. Rat cardiac muscle works well.
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


Not tested on cultured cells