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Permeabilization for IHC

Related

  • IHC guide: introduction
    • Tips for experimental design
        • IHC guide: sample prep
          • Tissue fixation, embedding and sectioning
            • Antigen retrieval
              • Permeabilization
                • Blocking
                    • IHC guide: immunostaining
                      • Choosing and optimizing primary antibodies
                        • Direct vs indirect detection
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                            • Chromogenic detection
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                                        • IHC guide: troubleshooting
                                            • IHC guide: controls

                                              ​Permeabilization is required when the antibody needs access to the inside of cells in order to detect the target antigen.

                                              Such antigens include intracellular proteins and cytoplasmic epitopes of transmembrane proteins. Solvents or detergents are typically used for permeabilization.

                                              Solvents can be used after fixation with crosslinking agent such as formaldehyde. Recommended for cytoskeletal, viral and some enzyme antigens.

                                              Detergents can either be harsh (eg Triton™  X-100 or NP-40) to disrupt proteins, or mild (eg Tween 20®, saponin or digitonin) so as to not dissolve plasma membranes. Detergent permeabilization can significantly improve antibody access to antigens in the cytoplasm, on the cytoplasmic face of the plasma membrane, and soluble nuclear antigens.

                                              Solvent and detergent guidelines


                                              SolventsComments
                                              Solvents

                                              Acetone            

                                                                                                                          

                                              Methanol

                                              Acetone fixation will also permeabilize

                                              Methanol fixation can be used to permeabilize but is not always effective

                                              Detergents

                                              Triton™ X-100 or NP-40

                                              Use 0.1 to 0.2% in PBS for 10 min only

                                              Tween 20®, saponin, digitonin and LeucopermUse 0.2 to 0.5% for 10 to 30 min

                                              >> Next page: blocking

                                              << Previous page: antigen retrieval

                                              Download the full IHC guide

                                              Tween 20® is a registered trademark of Croda Americas. Triton is a trademark of the Dow Chemical Company.

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