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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
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Name: Connexin 43 Antibody |
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Clone: Rabbit polyclonal |
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Supplier: Sigma-Aldrich |
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Catalog Number: C6219 |
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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. |
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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 |
Counterstain
| Reagent: Mayer's Hematoxylin |
| Staining Time: 30 seconds |
Results:
| 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 |
Notes:
Not tested on cultured cells |
References: