Tips for Step 10 – Incubate with Primary Antibody


  • Check the manufacturer’s datasheet to confirm that the antibody has been tested in IHC-paraffin. If the antibody has not been tested in IHC-paraffin but in another method such as IHC-frozen, do not assume that the antibody will automatically work in IHC-paraffin   

  • Consider using a polyclonal antibody when first establishing an IHC protocol. Although antigen retrieval is possible, the efficiency of the process is variable and certain epitopes might still remain inaccessible. Therefore a polyclonal antibody, which recognizes a multitude of epitopes due to its heterogeneous nature, provides a definite advantage over a monoclonal antibody which recognizes a single epitope. However, monoclonal antibodies have the advantage of batch-to-batch consistency and specificity, which often leads to lower background staining

  • Prior to performing the experiment, perform a literature search to determine the spatiotemporal localization of your protein of interest (in healthy and diseased tissue). For a preview of the expected staining pattern, the antibody datasheet or other web resources may also be consulted. Knowledge of the localization patterns also assists in selecting the optimal counterstain 

  • When using an antibody for the first time, always determine the optimal antibody dilution by titrating the antibody. You may also want to test different incubation periods and temperature conditions (4°C versus RT or 37°C) 

  • Should you encounter high background staining or suspect your primary antibody to be non-specific, consider including a concentration matched isotype control antibody (for example Mouse IgG2b Negative Control MCA691XZ) or pre-immune serum in your experimental design