antibody dose. two-photon microscopy, stroke, neurovascular unit == 1. Introduction == Two-photon laser scanning microscopy is an advanced imaging approach that enables high-resolution imaging of the cerebral microvasculature in animal models.1The activity of circulating immune cells is heavily regulated, and changes in this activity are an important indicator of disease. Increased leukocyte adhesion to the endothelium occurs in multiple neuroinflammatory conditions.2,3While histology is a useful technique, the dynamics of leukocyte circulation and adhesion requirein vivoobservation. Rhodamine 6G, a fluorescent dye, has been the standard marker for labeling leukocytesin vivofor two-photon imaging.4,5However, rhodamine 6G labels both leukocytes and platelets, and additional markers are required to differentiate between these cell types. In addition, rhodamine 6G has a wide emission spectrum, which makes it difficult to use together with other fluorophores.6,7There is a need for alternative leukocyte-specific markers that are accessible, long-lasting, and suitable for multichannel imaging experiments. CD45.2 is an allele variant of the transmembrane protein tyrosine phosphatase receptor type C (PTPRC, also Dodecanoylcarnitine known as CD45). CD45.2 is encoded by theallele and is present on the surface of all mouse leukocytes except in strains bearing the CD45.1 () allele, such as SJL mice.8The anti-mouse CD45.2 monoclonal antibody (clone 104) has been thoroughly validated as a murine pan-leukocyte marker for flow cytometry.9,10Clone 104 does not bind the CD45.1 allele. The function of CD45 is usually complex and divergent between mice and humans, with multiple splicing variant isoforms involved in human T cell differentiation.11Clinically, radioconjugates of anti-CD45 antibodies have been tested for bone marrow ablation for transplant preconditioning.12 To establish antibody dosage for optimizedin vivotwo-photon imaging of anti-CD45 labeled leukocytes, we first injected low, medium, and high concentrations of CD45.2 mAb intravenously (i.v.) and analyzed the fractions of labeled and saturated cells via flow cytometry. We then investigated potential for cell depletion and characterized its imaging parametersin vivoduring two-photon microscopy. We show that CD45.2 mAb fluorescent conjugates are usefulin vivoleukocyte marker for two-photon microscopy. Injection of CD45.2 mAb does not cause global leukocyte depletion. The antibody Dodecanoylcarnitine labels leukocytes specifically and with strong intensity ideal for observation of CD45. 2+ cells deep within the cortex. We also demonstrate that CD45.2 mAb are useful for detection of pathological leukocyte-endothelial interactions during venous stroke. == 2. Methods == == 2.1. Animals == Wild-type MUC12 BALB/c mice were purchased from Jackson Labs (#000651; Bar Harbor, Maine). Male and female mice aged 2 to 4 months were used for all experiments. Mice were socially housed five or less per cage in standard cages on a 12-h light-dark cycle, and those with cranial window implants were housed singly. All mice were monitored daily during the experimental period. Experiments were approved by the Seattle Childrens Research Institute Animal Use and Care Committee. == 2.2.In VivoAntibody Injection == Anti-mouse CD45.2 antibodies conjugated to APC, FITC, or Alexa 594 (clone 104, cat # 109814, 109805, or 109850, respectively; Biolegend) were injected at concentrations of 0.04, 0.4, orin a total volume of 50 to. Isotype control antibody (clone MOPC-173, cat # 400207; Biolegend) was used at. Antibodies were diluted in sterile saline and injected into the retro-orbital venous plexus. == 2.3. Blood Analysis == For flow cytometry analysis, blood was collected from the mouse facial vein by chin bleed 1 and 24 h after antibody injection [Fig. 1(a)]. Up toof blood was collected with this method. For complete blood count (CBC), a terminal blood draw of 200 towas taken from the Dodecanoylcarnitine inferior vena cava 24 h after antibody injection. Collected blood was immediately transferred into tripotassium ethylenediaminetetraacetic acid (K3EDTA)-coated blood collection tubes (95057-291, Greiner Bio-One) and kept at 4C until processing. CBC with differential counts of leukocyte subsets were measured on a Sysmex-XT-2000iV analyzer. == Fig. 1. == CD45.2 antibody labeling and Dodecanoylcarnitine saturation. (a) Schematic of experimental timeline. (b) Schematic of antibody.