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

Area of Science:

• Neuroscience
• Immunology
• Biomedical Engineering

Background:

• Studying leukocyte-endothelial interactions in vivo requires precise leukocyte labeling for two-photon microscopy of the cerebral microvasculature.
• Current techniques need optimization for clarity and specificity in living systems.

Purpose of the Study:

• To optimize dosing and imaging parameters for anti-CD45.2 monoclonal antibody (mAb) labeling of leukocytes.
• To evaluate the efficacy of this labeling technique in healthy mice and a microstroke model for in vivo two-photon microscopy.

Main Methods:

• BALB/c mice received retro-orbital injections of anti-CD45.2 mAb at varying doses.
• Flow cytometry assessed antibody saturation, while two-photon microscopy examined leukocyte labeling in the cortical microvasculature.
• A venous microstroke model was used to test labeling in pathological conditions.

Main Results:

• A dose of 0.4 mg/kg anti-CD45.2 mAb achieved 95% labeling of circulating leukocytes without causing depletion within 24 hours.
• Labeled leukocytes were visualized up to 150 µm deep from the cortical surface.
• The antibody successfully labeled rolling, crawling, and adherent leukocytes in venules near stroke-affected areas.

Conclusions:

• Anti-CD45.2 mAb is a reliable reagent for acute leukocyte labeling.
• This technique facilitates in vivo two-photon microscopy of the cortical microvasculature.
• It is applicable for studying leukocyte behavior in both healthy and pathological states, such as stroke.