Immunohistochemistry (IHC) has become an essential tool for clinical and translational research and diagnostic pathology. Intensity and tissue distribution of immunohistochemical staining have major impact on diagnosis and therapeutic opportunities in clinical pathology. Prognostic biomarkers, e.g. Ki67 expression in breast cancer (Yamamoto et al., 2013), provide information on the likely patient health outcome. Predictive biomarkers indicate the likely benefit of the patient from a specific treatment, e.g. Her2 overexpression and amplification are predictive of response to Trastuzumab (Hofmann et al., 2008). But also research projects are dependent on reproducible results.
Many tissue processing parameters have been identified to affect IHC-P staining results:
- Cold ischemia affects phosphoepitope stability in FFPE tissues (Bonnas et al., 2012)
- Fixatives influence tissue morphology and IHC staining results (Nietner et al., 2012)
- Inadequate fixation duration leads to decreased antigenicity (Werner et al., 2000).
SYSYs standard tissue preparation protocol comprises a 24 hrs fixation step in neutral buffered formalin. However, customers may use other fixation protocols. Therefore, we evaluated the influence of formalin fixation duration on immunohistochemical antigen detection in mouse spleen using three different time points: 13 hrs (presumed under-fixation), 24 hrs (standard fixation) and 48 hrs (presumed over-fixation). Eight HistoSure antibodies directed against common murine immune cell subtypes were tested, respectively.
Staining was performed according to the affiliated HistoSure IHC-P reference protocols. Staining intensity of 3-4 independent experiments was evaluated using a 4-point scoring system (Staining Intensity Score, SIS): 0 (no staining), 1 (light staining), 2 (medium staining) or 3 (dark staining).