Activation of neutrophils, eosinophils and macrophages during involution. (a) RNA expression for GRO1/IL-8/KC and LRG derived from microarray analysis. (b) RNA expression for BMAC, gp39, cathepsin S, MPS1, galectin 3 and CD68 derived from microarray analysis. The x-axes of both graphs describe the different developmental stages: virgin (V10, V12), pregnancy (P1, P2, P3, P8.5, P12.5, P14.5, P17.5), lactation (Lac1, Lac3, Lac7) and involution (Inv1, Inv2, Inv3, Inv4, Inv20). The y-axes describe the averaged scaled signal of the triplicate samples for each time point. The intensity data for LRG have been scaled down 10-fold for display. (c) Semi-quantitative RT–PCR for GRO1, LRG, BMAC and CD68 from a fourth independently collected RNA sample. (d) Identification of macrophages, neutrophils and eosinophils. The figure shows the immunohistochemical staining of a paraffin-embedded tissue section from a mouse mammary gland after 4 days of involution. Macrophages were identified by staining with F4/80 antibody and by their nuclear and cellular shapes. Eosinophils were identified by cross-reaction with the F4/80 antibody, polymorphonuclear structure and eosin staining. Neutrophils were identified through their polymorphonuclear structure, clear cytoplasm, slight eosin staining and non-staining with the F4/80 antibody. (e) Cell counts of neutrophils, eosinophils and macrophages present in the mammary tissue during involution. Tissue sections were stained with an antibody against F4/80 and counterstained with H&E; neutrophils and macrophages were counted. Cell counts were taken from 20 (neutrophils and eosinophils) and 50 (macrophages) high-power fields, respectively, from each section in triplicate from three individual mice.