Four hours before the intravital imaging, inject 20 milligrams per kilogram of LPS IP into a 16- to 20-gram 4 to 6-week-old male mouse to induce the mock bacterial inflammatory response.
Pre-warm a 0.9% saline solution in a 37-degree Celsius water bath for humidifying the plastic chamber and mesentery tissue at this time as well. Next, confirm the appropriate depth of anesthesia by a lack of response to toe pinch in the LPS-treated mouse, and then apply ointment to the animal's eyes.
Depilate the abdomen with a shaver, removing any loose hair with 70% ethanol-soaked gauze. Then, use a small curved forceps and scissors to open the abdomen.
Identify the epigastrical vessels, and open the peritoneum in the linea alba region to protect the vessels. Apply a few drops of pre-warmed saline into the abdominal cavity to keep the tissue moist, followed by a retro-orbital injection of 50 microliters of rhodamine 6G to label the circulating blood cells. Then, place the mouse in a 10-centimeter Petri dish, exteriorize a loop of ileum, and administrate the pre-warmed saline every other minute to keep the tissue moist.
To visualize the inflammatory response by intravital microscopy, place the mouse underneath the microscope and bring a mesentery vein with the diameter of 200 to 300 micrometers and as little as possible visible surrounding fat into the field of view.
Using the appropriate microscope software, record the blood cell-endothelial interactions for 1 minute in 4 different veins per mouse. Then, to analyze the cell-to-cell responses, confirm the stable and inter-individually blood flow conditions.
To quantify the number of rolling leukocytes, draw a vertical line through the vein, and manually count all of the leukocytes that cross this line in 1 minute. To determine the rolling velocity, draw two vertical lines through the vein 50 micrometers apart, and measure the time it takes a single leukocyte to cross between the lines while stably rolling on the endothelium.
To measure the leukocyte adhesion, draw a 200 by 200-micrometer square in the vein. Then manually count the firmly-adherent leukocytes that exhibit no visible movement within the square for 30 seconds. Finally, to quantify the platelet-leukocyte interactions, manually count the number of platelets bound to one leukocyte.
