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IHC guide v3 952x200px

Immunostaining in 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
                          • Secondary antibodies for IHC
                            • Chomogenic detection
                              • Substrates and chromogens
                                • Fluorescent detection
                                  • Counterstaining and special stains
                                    • Mounting
                                        • IHC guide: troubleshooting
                                            • IHC guide: controls

                                              ​

                                              The key to immunohistochemistry is the specific antibody-mediated detection of a target antigen, known as immunostaining or immunodetection.

                                              ​The choice of primary antibody is critical to the success of the IHC experiment. The most important feature of the primary antibody is its specificity for the target antigen. The antibody is then detected either directly, through a label that is directly conjugated to the primary antibody, or indirectly, using a labeled secondary antibody that has been raised against the host species and antibody type and subtype of the primary antibody. The antibody is visualized using either a fluorescent label or an enzyme that converts a soluble substrate into an insoluble chromogenic product.

                                              The steps in a typical immunostaining protocol are shown below. In direct immunostaining methods, the incubation step with the secondary antibody is omitted. The last step, where substrate is added, is omitted in fluorescent detection.


                                              IHC guide v3 immunostaining workflow 472px


                                              >> Next page: choosing and optimizing primary antibodies for IHC

                                              << Previous page: blocking

                                              Download the full IHC guide

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