To identify the leukocyte subpopulations present, normal corneal stromas were stained with antibodies to various leukocyte differentiation markers
(Fig. 1B) . The antibody CD11b, which detects complement receptor 3 (CR3) on monocytes, macrophages, dendritic cells, granulocytes, NK cells, and a subpopulation of T lymphocytes,
41 identified large numbers of cells in the stroma. Simultaneous two-color staining with CD45 and CD11b determined that 100% of the CD45
+ cells expressed CD11b
(Table 2) . The stroma also contained cells expressing the marker F4/80
(Fig. 1C) , a glycoprotein that is specifically restricted to subsets of macrophages, monocytes and dendritic cells.
42 43 There was a wide variation noted in the surface intensity of F4/80 on cells. Some cells were quite brightly stained, whereas others expressed very low levels of this antigen. The distribution of F4/80-reactive cells in the corneal stroma was less uniform than the distribution of the total CD45/CD11b
+ population. The F4/80
+ cells tended to occur in clusters of relatively high density, whereas other portions of the same stroma contained fewer positive cells. There was no apparent radial gradient of F4/80 expression between the periphery and the center. Overall, approximately 52% ± 12% of leukocytes in the corneal stroma were F4/80
+, as determine by combined staining with CD45 and F4/80
(Table 2) . That all the cells in the stroma reacted with CD11b and many reacted with F4/80 suggests that most of the cells probably belonged to either the macrophage/monocyte or dendritic cell lineage. These two populations were distinguished by staining corneas with antibody to the integrin CD11c, which in the mouse is expressed on all dendritic cells.
43 44 45 46 However, only rare CD11c
+ cells (<20 cells per cornea) were observed in the pericentral or central stroma
(Fig. 1D) . Some CD11c
+ cells were observed at the limbus and peripheral edge of the cornea, consistent with previous reports describing dendritic cells in this area. As a positive control, corneal stromas from mice with inflamed HSV-1-infected corneas (day 14 after infection) were reacted with the CD11c antibody. In contrast to normal corneas, large numbers of CD11c
+ dendritic cells were readily observed in the pericentral region of infected corneas
(Fig. 1E) . The slight blur to the images shown is due to the tyramide amplification process used to aid in visualizing CD11c antibody staining. Thus, it appears that dendritic cells represent only a very minor portion of the leukocytes that are resident in the normal mouse corneal stroma. Antibody to the pan-granulocyte marker Ly-6G
47 occasionally detected a scattered grouping of small numbers of cells in the pericentral stroma, but the overall density per corneal stroma was lower than 25 cells per mm
2 (data not shown). T lymphocytes and NK cells were completely absent from the normal corneal stroma, in that no staining was observed with antibodies to the pan-T-cell marker CD3
48 or the pan-NK cell marker DX5
49 (data not shown). As a positive control for these antibodies, we examined HSV-1-infected corneas. Numerous CD3
+ cells were detected in corneas 14 days after HSV-1 infection. We were also able to image DX5
+ cells specifically in unfixed corneas at 3 days after HSV-1 infection (data not shown, see the Methods section for techniques used). Taken together, the data indicate that most of the cells detected in the normal corneal stroma were myeloid in lineage.