FIGURE 2.

α_Dβ₂ is expressed by distinct populations of macrophages in bone marrow, thymus, and spleen. Frozen sections of freshly isolated tissues were stained with mAb 205c using an immunoperoxidase detection method. Tissues from 8- to 12-wk-old C57BL/6 mice were frozen in Tissue-Tek (Sakura Finetek) and 8-μm cryostat sections were deposited on Superfrost Plus slides (Fisher Scientific). The sections were then fixed in cold acetone for 10 min. Endogenous tissue peroxidase was eliminated by incubation with 0.01% hydrogen peroxide for 15 min. The sections were blocked by incubating with 5% normal goat serum, 1% BSA and avidin solution (Vector Laboratories) for 30 min, and primary Abs (hamster mAbs 205c or 279f raised against mouse α_D) were applied to the slides and incubated overnight at 4°C. A secondary Ab (biotin-conjugated goat anti-hamster; Jackson ImmunoResearch Laboratories) was applied at a 1/1000 dilution and incubated for 30 min. Negative controls were performed by omitting the primary Ab or by applying an irrelevant isotype-matched primary Ab as a control. ABC reagent was prepared according to the manufacturer’s instructions (Vector Laboratories) and applied to the sections for 30 min. Staining was developed using a NovaRed kit (Vector Laboratories), followed by counterstaining with Gill’s hematoxylin, dehydration, and mounting using Cytoseal 60 medium (VWR). In each pair of figures, the panel on the left is magnified ×40 and on the right ×800. These distributions of α_D are representative of multiple studies. A and B, Bone marrow; C and D, thymus; E and F, spleen; WP, white pulp; RP, red pulp.