Sequential Histochemical Staining for Resident and Recruited Macrophages

A new histochemical technique is described that permits differentiation of resident from recruited macrophages by staining of paraffin sections of tissues from rats and mice. Resident macrophages are identified by their ability to phagocytose and retain intravenously injected colloidal Prussian blue. New macrophages that emigrate into tissue are identified by phagocytosis of a second colloid, iron dextran. Paraffin sections of formalin‐fixed tissues are sequentially stained for the presence of the two colloids with different chromogens, the endogenous pseudo‐peroxidase activity of colloidal Prussian blue used to catalyze the polymerization of diaminobenzidine and after conversion of iron dextran to Prussian blue, the second colloid used to catalyze the polymerization of tetramethylbenzidine. The staining results in resident macrophages staining brown while newly recruited macrophages stain blue. The studies have shown that colloidal Prussian blue is stable in vivo and neither loses its catalytic activity nor undergoes extensive redistribution. They also show that the technique can be used to measure Kupffer cell recruitment stimulated by complete Freund's adjuvant in rats and tumor‐associated macrophage recruitment in subcutaneous and spontaneous liver metastases in mice.