Figure 2.

Technical considerations for intravascular staining. (a–d) Thy1.1 + P14 CD8 T cell chimeras (a), CD45.1 + SMARTA CD4 T cell chimeras (b) or CD45.2 + C57BL/6 mice were infected with LCMV i.t. (c,d). (a–g) After 12–15 d, mice were injected i.v. with the indicated mAb (clones are in parentheses), killed 3 min later and lymphocytes were either isolated and further stained ex vivo for flow cytometric analysis (a–d) or tissue sections were examined by epifluorescence microscopy (e–g). (a–d) Plots are gated on CD8P+ and Thy1.1 + (a), CD4 (clone RM4–5) + and CD45.1 + (b), CD45.2 + (clone 104, stained ex vivo), CD8α+ and H2-D^b/gp_33–41 MHC I tetramer ⁺ (c) or CD8α⁺ (stained ex vivo) and H2-D^b/gp_33–41 MHC I tetramer ⁺ lymphocytes (d). Plots are representative of at least three experiments and nine mice per condition. (e–g) mAb specific for collagen type IV (green) and CD8β or CD4 (cyan, as indicated) stained ex vivo, and the indicated i.v. injected mAb (red) on spleen sections. Images are representative of at least three experiments and eight mice per condition. Scale bars, 100 µm. (h) At 30 d after i.t. LCMV infection of C57BL/6 mice, anti-CD45.2 i.v. mAb staining intensity was examined on CD8α ⁺ H2-D^b/gp_33–41 MHC I tetramer ⁺ lymphocytes isolated from blood (PBL, red), spleen (blue) and lung (black). Representative of three experiments totaling nine mice.