Selective inhibition of nonspecific eosinophil staining or identification of eosinophilic granulocytes by paired counterstaining in immunofluorescence studies.

Nonspecific staining of eosinophilic granulocytes produced by nonimmune rabbit IgG conjugates, highly labeled with fluorescein isothiocyanate (FITC) or tetramethylrhodamine isothiocyanate (MRITC), was efficiently inhibited by pretreatment of tissue sections with diaminobenzidine and hydrogen peroxide; the brown reaction product developed in the presence of this substrate by the endogenous peroxidase of the granulocytes sheltered these cells from subsequent nonspecific staining without interfering with specific immunofluorescence staining of other tissue elements. Similar results were obtained with the alternative noncarcinogenic peroxidase substrate, p-phenylenediamine-pyrocatechol. Alternatively, the eosinophils could be identified by their nonspecific staining with FITC-labeled bovine serum albumin when this conjugate was combined with an MRITC conjugate used for specific staining; optical conditions selective for green and red fluorescence discriminated distinctly between double-stained eosinophils and purely red specifically stained cells.