Identification of β2 Integrins in Bovine Neutrophils by Scanning Electron Microscopy in the Backscatter Mode and Transmission Electron Microscopy

Neutrophils (I x 105) were suspended in phosphate-buffered saline (PBS) and I% bovine serum albumin (BSA) for 10 minutes at 37 C to block nonspecific binding sites. Cells were centrifuged (900 rpm for 3 minutes) and rinsed twice with PBS (pH 7.2). Some of the cell suspensions were incubated with platelet activating factor (PAF; 100 ng/ml) for 5 minutes. Neutrophils were incubated with the optimal concentration of primary antibody (RI5.7; 5 f.Lg/ml final concentration) for I hour, rinsed, resuspended, and then exposed to the secondary antibody conjugated to 30-nm gold (I: 20 dilution final dilution) for 30 minutes. To assess possible nonspecific binding, control preparations used neutrophils with normal CD 18 expression but that either lacked the primary antibody or were exposed to a mouse antibody specific for unrelated antigens. Neutrophils were rinsed three times, fixed with I% glutaraldehyde (pH 7.4) for I hour, and rinsed again. A single layer of fixed neutrophils was applied to a round glass coverslip precoated with fresh poly-r.-Iysine and mounted with aluminum paint onto an aluminum stub; remaining neutrophils were processed for transmission electron microscopy. The preparation was dehydrated in graded alcohols, dried in a critical point dryer (Balzers CPO 020, Liechtenstein), coated with carbon (l0-20-nm layer), and examined on a JEOL scanning electron microscope (Tokyo, Japan) equipped with a backscatter detector. Gold sputter coating and osmium fixation could not be used because these heavy metals would cause haphazard and unorganized electron reflection and obliterate the colloidal gold particles. Carbon (atomic weight = 14), however, is light enough to coat the samples without interfering with the electroreflective properties of the gold particles and prevents undesirable charging effects. The optimal magnification settings were 18,000 x magnification and 15 kV. Gold particles were present on the surface of neutrophils from cows that expressed normal levels of CD 18. These foci were widely separated by 0.025-0.05 f.Lm (Fig. I) and were present on both the tips of surface pseudopodia and along the deeper recesses of the plasma membrane. The particles were easily visible in the backscatter (BE) mode but were undetectable in the conventional scanning mode. For quantitative work, five random cells per stub were photographed (18,000 x ) from three replicate tests, and gold particles were counted. By scanning electron microscopy, the number of gold particles averaged 62.0 and 51.4 particles per field of