Antibody-based in vivo leukocyte label for two-photon brain imaging in mice

Significance: To study leukocyte-endothelial interactions in a living system, robust and specific leukocyte labeling techniques are needed for in vivo two-photon microscopy of the cerebral microvasculature. Aim: We tested fluorophore-conjugated anti-CD45.2 monoclonal antibodies (mAb) to optimize dosing and two-photon imaging parameters for leukocyte labeling in healthy mice and a venous microstroke model. Approach: We retro-orbitally injected anti-CD45.2 mAb at 0.04, 0.4, and 2 mg / kg into BALB/c mice and used flow cytometry to analyze antibody saturation. Leukocyte labeling in the cortical microvasculature was examined by two-photon imaging. We also tested the application of CD45.2 mAb in a pathological leukocyte-endothelial adhesion model by photothrombotically occluding cortical penetrating venules. Results: We found that 0.4 mg / kg of anti-CD45.2 antibody intravenously was sufficient to label 95% of circulating leukocytes. There was no depletion of circulating leukocytes after 24 h at the dosages tested. Labeled leukocytes could be observed as deep as 550 μm from the cortical surface. The antibody reliably labeled rolling, crawling, and adherent leukocytes in venules around the stroke-affected tissues. Conclusion: We show that the anti-CD45.2 mAb is a robust reagent for acute labeling of leukocytes during in vivo two-photon microscopy of the cortical microvasculature.