Alexa fluor 594 labeled anti cd3 antibody

Alexa Fluor 594–labeled anti-CD3 antibody (BioLegend) and Alexa Fluor 488–labeled anti-CD44 antibody (BioLegend) were used to detect activated memory T cells and directly and quantitatively visualize their glomerular intravascular homing in C57BL/6, NZM WT, and NZM.BAFFTg mice (3–4 weeks old) using serial MPM. These antibodies were administered via retro-orbital sinus each at a dose of 30 μL. Clearly positively labeled cells could be seen immediately after antibody injection. The numbers of CD3 and CD44 double-positive cells in entire glomeruli were calculated in 3D volume (xyz) images before and after H treatment. Multiple glomeruli per each mouse were analyzed as indicated to avoid sampling errors. Positively labeled cells could be identified in skin, muscle, peritoneal membrane, and spleen and enumerated in single xy images before and after H treatment.

Alexa Fluor 594–labeled anti-CD3 antibody (BioLegend) and Alexa Fluor 488–labeled anti-CD44 antibodies (BioLegend) were used to detect activated T cells and to directly and quantitatively visualize their glomerular intravascular homing in WT and AS mice using time-lapse MPM as described recently (10 (link)). These antibodies were administered via retro-orbital sinus each at dose of 30 μl. Positively labeled cells could be seen clearly immediately after antibody injection. The number of CD3 and CD44 double-positive cells in entire glomeruli were counted in 3D volume (xyz) images before and after H treatment. Multiple glomeruli per each mouse were analyzed to avoid sampling errors. In some experiments, time-lapse imaging of the same z plane was performed for 5–10 minutes, and single projection images of the entire xyt sequence were generated to illustrate the dynamics and magnitude (CD3⁺CD44⁺ cell density) of immune cell surveillance.