M. Fero 1/8/09

Procedure

1. Register online
2. Bring mouse in clean cage to imager
3. Sign in on log sheet
4. Clean inside of Xenogen system and external mouse anesthesia chamber. To minimize background, use black plastic background sheet for fluorescence, use black paper for luminescence.
5. Anesthesia
   1. Turn on gas cylinder (but don't change gas regulator on cylinder)
   2. On Xenogen box - turn 'Gas' knob to 'on' (always leave power button on).
      1. On anesthesia machine:
         1. O2: On
         2. Vaccuum: On
         3. Switch up (open) valves to Chamber and IVIS. Set flow rate with ball valve to midway (~2 L/min).
         4. Set guage on lid of Isofluorane container to '2' (can change to alter level of anesthesia).
         5. Place mice in chamber.
6. Login to computer
7. Living Image #3 software
   1. Launch program (icon on Desktop). Enter your initials to tag images.
   2. Control Panel: Initialize IVIS System
   3. Choose field of view (A-D) depending on # of mice to image simultaneously.
8. Position animals: (Alternatively the plastic phantom mouse can be used with a fluorescent insert).
   1. Open door: Green laser rectangle should appear
   2. Place nose cones / corks in manifold and position at back of green rectangle
   3. Transfer mice from external chamber to IVIS nose cones. Turn off gas valve to chamber.
9. Acquire images:
   1. IVIS Acquisition Control Panel
      1. Select Fluorescence vs. Luminescence
      2. Exposure time: default = 1 sec.
      3. Binning: default = medium (4 pixels per bin). Decrease if saturated, increase if very faint.
      4. f/stop: default = 2
      5. Excitation/Emission: Set according to fluorochrome. GFP/FITC = preset #3? (see phantom mouse booklet)
      6. Select 'Acquire Image'
   2. Image Window:
      1. Counts should be between 600 and 600k? (or 60k?)
      2. Change units from Counts to Efficiency (to compensate for increased efficiency at center of image)
   3. Multi-sequence: In Control Panel select 'Sequence setup'.
      1. Set delay time, wavelength, exposure, etc.
      2. Acquire images
      3. In image window select 'display all' icon (upper right) to manipulate individual images.
10. Image analysis (can be done post hoc with Living Image software in computer facility)
    1. Set ROI and filters to quantify regions of interest.
11. Save images to desktop in Fero Lab folder. Map Fred network drive and transfer your files. Then disconnect Fred.
12. Cleanup
    1. Return mice to cage
    2. Turn off Isofluorane. Flush for 1 min. then turn off gas flow on anesthesia machine and gas cylinder (opposite of 5.3, above).
    3. Use a paper towel moistened with Clidox to clean all surfaces that have been in contact with mice.
       1. Inside Xenogen chamber, Plastic backing, nose cones, door handle.
       2. External anesthesia chamber.
          Area around mouse cage.
13. Sign out on log sheet. Return mice to their proper room and racks.

Tags: Fero-Lab-Protocols, Fero-Mice