IHC Troubleshooting: High Background

The good immunohistochemical staining is a foundation of correct judgment for staining, since there are many steps and links through the whole immunohistochemistry process, each step and links are likely to affect the final result of the staining, so to do an immunohistochemistry quality slice is not a very easy thing.

Background may occur in the control and specimen tissue. In some organizations have seen a significant background staining, such as connective tissue, adipose tissue and epithelium. There are a variety of reasons resulting for high background. Summarized as follows

  Possible reasons Solutions
1 Antibody incubation time was too long Decrease the antibody
2 Polyclonal primary antibody Try a monoclonal primary antibody
3 Serum blocking was absent or insufficient Increase the blocking time
4 High antibody concentration Optimize the antibody concentration
5 Presence of endogenous Block endogenous peroxidase activity properly
6 Insufficient PBS washing Wash with PBS for long enoug and for enough times to eliminate fixatives