In situ cytokine expression in LCH biopsies. Single immunohistochemical labeling was used to assess cytokine production in frozen sections of LCH biopsies. (A) IL-1 demonstrated in a positive control tissue sample from human tonsil. Such control tissue was included on each glass slide to internally validate different cytokine stainings (original magnification × 100). (B) Human tonsil: negative control staining using an isotype-matched primary antibody of irrelevant specificity to exclude nonspecific binding of secondary and tertiary step reagents (original magnification × 100).8(C) IL-1–producing cells in a LCH lesion in bone (patient no. S926704). Note the high density of cytokine-expressing cells and the very intense staining of most of the cytoplasm, compared with (B) (original magnification × 100). (D) IL-3–producing cells in a monoostotic lesion (patient no. S9411100; original magnification × 100). (E) IL-10–producing cells in a affected lymph node in disseminated LCH (patient no. S93292). Note that no clustering occurs and that isolated cells display only partially cytokine-filled cytoplasm (original magnification × 100). (F) GM-CSF–producing cells in a monoostotic lesion (patient no. 9408P025; original magnification × 50).