File:Control-of-Uterine-Microenvironment-by-Foxp3+-Cells-Facilitates-Embryo-Implantation-Movie 1.ogv

English: Estrus: 3D reconstruction of live 2-photon-microscopy of uterus from a representative Foxp3gfp animal during the estrus phase of the estrus cycle. The green cells are Tregs clustered at the mesometrial region. Animals were anesthetized by i.p. injection of ketamine and xylazine, 120 or 16 μg/g of mouse weight, respectively, and kept on a heating pad at 37°C. One of the uterine horns was carefully exposed and 0.1 M caffeine (Sigma-Aldrich, USA) was applied to decrease uterine contractions. For maternal blood visualization, animals were intravenously injected with 100 μl of Rhodamine B isothiocyanate-Dextran (RhoB-Dex) 70,000 KDa (Sigma-Aldrich, Inc., USA) before acquiring images in a multiphoton laser scanning microscope (MPLSM). We used a Prairie Ultima 2-photon microscope (Prairie Technologies, Inc.). The microscope was equipped with a Chameleon Ti:Sapphire laser (Coherent, Inc., USA), four top PMTs for simultaneous up to four channel acquisitions and a 20× water immersion objective (Olympus, Inc., USA). The laser was tuned to 880 nm to allow for concomitant excitation of RhoB-Dex and GFP+-Treg. The wavelength emission for RhoB-Dex and GFP is 590 and 509 nm, respectively. Sequential images were acquired for observation of Treg in the uterus. For analysis of uterine Treg images, we have developed our own software algorithms based on endogenous tissue markers information (e.g., location of blood vessels) from consecutive z-stacks acquisitions for stabilization of Treg movies. Once the images were stabilized, we used Imaris software (Bitplane AG, Inc.) for reconstruction of three-dimensional models in order to determine distribution in the uterus.