Immunohistochemical Enumeration of Mast Cells in CML

The indirect immunoperoxidase staining technique was performed with serial sections (2 μM) of formalin-fixed and paraffin-embedded BM as described [49 (link)-51 (link)]. For MC detection and enumeration, antibodies against KIT (CD117) (polyclonal) and tryptase (mAb G3) were applied overnight. Slides were then washed and incubated with biotinylated anti-rabbit IgG or anti-mouse IgG, washed, and then exposed to streptavidin-biotin-peroxidase-complex. AEC was used as chromogen. The numbers of tryptase+ cells and KIT+ cells (MC) were determined using an Olympus BX50F4 microscope connected to a DP21 camera (Olympus, Hamburg, Germany) and expressed as percent of nucleated BM cells. We also confirmed the presence of MC by Giemsa staining and counted the numbers of MC on Giemsa-stained BM sections in our CML patients.