1. Why is 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide (EDAC) important as fixative?

The coupling with this bifunctional fixative is effective at neutral and/or slightly acidic conditions. Use of EDAC results in highly sensitive and specific staining, which allows dilution of the antisera maximally. The antisera do not recognize antigens coupled to tissue proteins with aldehydes with sufficient sensitivity and specificity.

2. Is EDAC suitable as a fixative for other antisera we would like to use for double or triple staining?

Many antisera work well on EDAC-fixed tissues. However, e.g. paraformaldehyde can be used to postfix the tissue to improve staining with other antisera.

3. What are the ciritical issues in fixation of tissues with EDAC?

The EDAC solution should be prepared just before use. The pH is critical (preferable pH 6.9-7.1). If tissues are immersed rather than perfused, the solution can be changed to a fresh one during extended fixation.