American Society of Hematology

Fig. 2.

$Fig. 2. Electron micrographs of frozen thin sections from fractions containing azurophil granules, section of fractions # 2 (a and c), and specific granules # 9 (b and d). (a) Frozen thin section of fraction #2, which is labeled for Man 6-P GP with biotinylated probe, and detected with streptavidin gold-10. Most of the organelles in fraction #2 appear to be azurophilic granules labeled for Man 6-P GP (Ag). Some of the granules have extracted contents (Ag), while others retain their content (Ag′). This variation in content preservation has been previously observed in intact cells. (b) Fraction #10 contained typical specific granules (Sg) negative for Man 6-P GP. Indeed, compared to (a), only a few gold particles were present, and they were in MLC (arrow) and rare granular structures. Fractions 2 (c) and 10 (d) were labeled for LAMP-2 with 10 nm gold. Fraction 2 consisted mostly of azurophilic granules (Ag and Ag′); these granules were devoid of immunoreactivity (c). Most of the organelles in fraction 10 appear to be specific granules (Sg), which are not labeled, whereas the gold labeling is present in MLC (arrows). Tissue preparation for figures of electron micrographs, except 8: Fractions or cells were fixed in 2% paraformaldehyde, 0.05% glutaraldehyde for 1 hour at 4°C embedded in sucrose, frozen and processed for ultrathin-cryosectioning. For Fig 8b and c: leukocytes were fixed in 4% paraformaldehyde-lysine, using the buffers of McLean and Nakane.35$

Electron micrographs of frozen thin sections from fractions containing azurophil granules, section of fractions # 2 (a and c), and specific granules # 9 (b and d). (a) Frozen thin section of fraction #2, which is labeled for Man 6-P GP with biotinylated probe, and detected with streptavidin gold-10. Most of the organelles in fraction #2 appear to be azurophilic granules labeled for Man 6-P GP (Ag). Some of the granules have extracted contents (Ag), while others retain their content (Ag′). This variation in content preservation has been previously observed in intact cells. (b) Fraction #10 contained typical specific granules (Sg) negative for Man 6-P GP. Indeed, compared to (a), only a few gold particles were present, and they were in MLC (arrow) and rare granular structures. Fractions 2 (c) and 10 (d) were labeled for LAMP-2 with 10 nm gold. Fraction 2 consisted mostly of azurophilic granules (Ag and Ag′); these granules were devoid of immunoreactivity (c). Most of the organelles in fraction 10 appear to be specific granules (Sg), which are not labeled, whereas the gold labeling is present in MLC (arrows). Tissue preparation for figures of electron micrographs, except 8: Fractions or cells were fixed in 2% paraformaldehyde, 0.05% glutaraldehyde for 1 hour at 4°C embedded in sucrose, frozen and processed for ultrathin-cryosectioning. For Fig 8b and c: leukocytes were fixed in 4% paraformaldehyde-lysine, using the buffers of McLean and Nakane.35

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