Mice were anaesthetized with Ketamin /Xylazine (65/5 mg/kg bwt, i.p.), and an aqueous solution of luciferin (150 mg/kg intraperitoneally) (Xenogen Corp.) was injected 5 minutes prior to imaging. The live anesthetized mice were imaged using the IVIS200 in vivo imaging system (Xenogen Corp.). A grayscale body surface reference image (digital photograph) was taken under weak illumination. After switching off the light source, photons emitted from luciferase-expressing cells within the animal body and transmitted through the tissue were quantified over a defined period of time ranging up to 5 minutes using the software program "Living Image" (Xenogen Corp.) as an overlay on Igor (Xenogen Corp.). Raw values are reported as photons/second/cm2/sr.
Some Specific Protocols:
Continuous Monitoring of in vivo Gene Expression: During a 6-day continuous observation, the transfected luciferase gene as shown in bioluminescence images (blue-red spot on the top of mouse head) can be detected as soon as 24 hours after initial gene transfection.
Confirming NOX4 and Luciferase Expression in PAG: The top two panels of images present the non-invasive bioluminescent signal on the skull surface of animals in both groups: Luciferase only and Luciferase with NOX4. Here, luciferase only group was considered the control group since it is only a reporter gene. The bottom two ex vivo images show the dissected brain from one animal in each group, which confirmed that the signal on animal skull surface was derived from PAG area transfected with this reporter gene.
Spectral Analysis Shows the Depth of Signal: The depth of bioluminescent reporters from the surface were quantitated, which is related to the luciferase emission spectrum (the left bottom curve) and the tissue optical properties (the right bottom curve). As shown on the left, the calculated value for the depth of luciferase reporter detected was about 2.2 mm, which was around the depth of i.c.v. injection. This further confirmed that the developed signal on the skull surface was propagated from the PAG area.