Mouse spleen tissue as a staining intensity reference for immunohistochemistry

Immunohistochemistry (IHC) is widely used in diagnostic practice and research, but it is limited due to its subjective nature and weakness in reproducibility. For successful interpretation, IHC requires an internal reference system that controls for procedural variables and provides a staining intensity reference. We investigated the feasibility of using mouse spleen tissue as an intensity reference in conventional IHC. Formalin-fixed, paraffin-embedded mouse (BALB/c) spleen tissue was stained with variable procedural conditions including primary antibody (Ab) types, antigen retrieval methods, chromogen exposure times, and secondary Ab concentrations. Mouse spleen tissue showed identical staining intensity regardless of primary Ab types, even without primary Ab, and showed minimal differences according to retrieval methods. However, it showed various staining intensities according to chromogen exposure time and secondary Ab concentration. When mouse spleen was included in tissue microarrays and compared with the c-erbB2 IHC scoring system, splenic B cells showed weak membrane staining compatible with score 1+, whereas splenic plasma cells showed strong staining intensity compatible with score 3+. These results show that mouse spleen tissue can serve as a staining intensity reference for the interpretation of IHC.