Posted on July 5th, 2016 by UNM CC
M. Fero 1/8/09
- Register online
- Bring mouse in clean cage to imager
- Sign in on log sheet
- 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.
- Turn on gas cylinder (but don't change gas regulator on cylinder)
- On Xenogen box - turn 'Gas' knob to 'on' (always leave power button on).
- On anesthesia machine:
- O2: On
- Vaccuum: On
- Switch up (open) valves to Chamber and IVIS. Set flow rate with ball valve to midway (~2 L/min).
- Set guage on lid of Isofluorane container to '2' (can change to alter level of anesthesia).
- Place mice in chamber.
- Login to computer
- Living Image #3 software
- Launch program (icon on Desktop). Enter your initials to tag images.
- Control Panel: Initialize IVIS System
- Choose field of view (A-D) depending on # of mice to image simultaneously.
- Position animals: (Alternatively the plastic phantom mouse can be used with a fluorescent insert).
- Open door: Green laser rectangle should appear
- Place nose cones / corks in manifold and position at back of green rectangle
- Transfer mice from external chamber to IVIS nose cones. Turn off gas valve to chamber.
- Acquire images:
- IVIS Acquisition Control Panel
- Select Fluorescence vs. Luminescence
- Exposure time: default = 1 sec.
- Binning: default = medium (4 pixels per bin). Decrease if saturated, increase if very faint.
- f/stop: default = 2
- Excitation/Emission: Set according to fluorochrome. GFP/FITC = preset #3? (see phantom mouse booklet)
- Select 'Acquire Image'
- Image Window:
- Counts should be between 600 and 600k? (or 60k?)
- Change units from Counts to Efficiency (to compensate for increased efficiency at center of image)
- Multi-sequence: In Control Panel select 'Sequence setup'.
- Set delay time, wavelength, exposure, etc.
- Acquire images
- In image window select 'display all' icon (upper right) to manipulate individual images.
- Image analysis (can be done post hoc with Living Image software in computer facility)
- Set ROI and filters to quantify regions of interest.
- Save images to desktop in Fero Lab folder. Map Fred network drive and transfer your files. Then disconnect Fred.
- Return mice to cage
- Turn off Isofluorane. Flush for 1 min. then turn off gas flow on anesthesia machine and gas cylinder (opposite of 5.3, above).
- Use a paper towel moistened with Clidox to clean all surfaces that have been in contact with mice.
- Inside Xenogen chamber, Plastic backing, nose cones, door handle.
- External anesthesia chamber.
Area around mouse cage.
- Sign out on log sheet. Return mice to their proper room and racks.