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

Autor:	Faulhaber LD; Center for Developmental Biology and Regenerative Medicine, Seattle, Washington, United States.; Seattle Children's Research Institute, Center for Integrative Brain Research, Seattle, Washington, United States., D'Costa O; Seattle Children's Research Institute, Center for Integrative Brain Research, Seattle, Washington, United States., Shih AY; Center for Developmental Biology and Regenerative Medicine, Seattle, Washington, United States.; University of Washington, Department of Pediatrics, Seattle, Washington, United States.; University of Washington, Department of Bioengineering, Seattle, Washington, United States., Gust J; Seattle Children's Research Institute, Center for Integrative Brain Research, Seattle, Washington, United States.; University of Washington, Department of Neurology, Seattle, Washington, United States.
Jazyk:	angličtina
Zdroj:	Neurophotonics [Neurophotonics] 2022 Jul; Vol. 9 (3), pp. 031917. Date of Electronic Publication: 2022 May 24.
DOI:	10.1117/1.NPh.9.3.031917
Abstrakt:	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    μ m 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. (© 2022 The Authors.)
Databáze:	MEDLINE
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