The detection of CD2+, CD4+, CD8+, and WC1+ T lymphocytes, B cells and macrophages in fixed and paraffin embedded bovine tissue using a range of antigen recovery and signal amplification techniques

Abstract

In order to develop procedures to label the main bovine leucocyte populations in paraffin embedded sections, the immunoreactivity of 25 monoclonal antibodies (mAbs) to different leucocyte antigens was assessed with formal dichromate (FD5) and 10% formalin fixation, a battery of antigen retrieval (AR) methods, and the biotin-tyramide amplification system. All the leucocyte populations investigated (CD2+, CD4+, CD8+, WC1+ T lymphocytes, B cells and macrophages) were strongly and specifically detectable under an appropriate combination of mAb, AR method and signal amplification system. CD4 and CD8 required the most stringent conditions and could only be demonstrated in FD5 fixed sections. For detection of CD2, WC1+ T lymphocytes, B cells and macrophages, all the mAbs produced immunoreactivity in FD5 or formalin fixed tissues. The need to check a range of different AR methods is stressed, as the method of choice varied for each individual mAb. The incorporation of the signal amplification system was necessary to observe a strong signal and the complete distribution of CD4, CD8 and B cells. Fixation by FD5 proved to be better than formalin for the preservation of surface antigens but it was inferior for the detection of markers which were found to show cytoplasmic immunoreactivity, such as the macrophage marker MAC387 or the B cell markers BAQ155 or IL-A59.