Antibodies against mPR3 generated in mPR3/mNE-deficient mice recognize mPR3 from mouse neutrophils. (A) Western blot analyses of murine neutrophil lysates derived from wild-type mice (lanes 1-2), knockout mice lacking mPR3 (lanes 3-4), and of recombinant mPR3 (lanes 5-6) resolved by SDS-PAGE under reducing (lanes 1, 3, and 5) and nonreducing conditions (lanes 2, 4, and 6) using antiserum pool 1; molecular mass markers are given on the left. Murine antibodies recognize mPR3 in wild-type neutrophils, but do not stain proteins in neutrophil lysates of mPR3/mNE-deficient mice (mPR3/mNE-def). The faint band of approximately 20 kDa in lanes 5 and 6 is a minor degradation product of mPR3 as determined by N-terminal amino acid sequencing. Equivalent results were obtained with antiserum pool 2 (not shown). (B) Indirect immunofluorescence microscopy using PFA-fixed murine neutrophils from wild-type and mPR3/mNE-deficient mice as a substrate and mPR3-specific antiserum pool 1 to demonstrate a PR3-ANCA–like pattern. The cytoplasm of wild-type granulocytes is stained with strong granular accentuation. Equivalent results were obtained with PR3 antiserum pool 2, whereas neither mock immune serum-treated wild-type neutrophils nor PR3 antiserum-treated neutrophils from mPR3/mNE-deficient mice stained positively (not shown). The fixed cells were embedded in Movid (Calbiochem, CA). Microscopy and photography were performed with an Axioplan 2 microscope (Zeiss, Göttingen, Germany) equipped with a 10 × /2.5 ocular, 20 × /0.5 neofluar, 40 × /0.75 neofluar, and 63 × /1.4 oil (inset) objectives, and a SpotRT color digital camera (Diagnostic Instruments, Sterling Heights, MI) Fluorescence images were electronically recorded with Metamorph software (Visitron Systems, Puchheim, Germany). Original magnification × 400; inset × 630.