Fv1000

The fluorescently stained tissues on hand, forearm, upper arm, axilla and thorax were sampled, respectively. Sampled tissues were examined histologically by frozen fluorescence, Elastic van Gieson or H&E staining according to standard procedures. Chinese ink was used to permanently locate the exact position of fluorescence in the tissue samples. After the sampling, frozen cross‐sections of 5‐10 μm in thickness were cut from the tissues containing the fluorescent pathways and observed under a fluorescence microscopy. The same slides of the frozen sections were then examined using haematoxylin & eosin (HE) staining and the combined staining methods of Elastic van Gieson (VG), respectively.
The tissues used were the same samples as those of the frozen sections. Two‐photon laser scanning microscopy (TPLSM, Olympus FV1000 and Spectra‐Physics Mai Tai DeepSee) was employed to exam all samples obtained from the skins, and the blood vessels including their surrounding connective tissues. TPLSM was performed at 960 nm for excitation and 495‐630 nm for emission. For each sample, 25 slices were captured using a 0.1‐μm interval at a 512 × 512 resolution.

Animals were anesthetized via i.p. injection of ketamine (100 mg kg⁻¹ body weight) and xylazine (10 mg kg⁻¹ body weight), and a 3-mm cranial window was implanted over the somato‐sensory cortex of 12–14-week-old Irf8^+/+, Irf8^+/− and Irf8^−/− mice. During the imaging session, the body temperature was monitored and maintained at 36–37 °C using a heating blanket. Depth of anesthesia was assessed by monitoring pinch withdrawal and vibrissae movements.
Laser lesion and acute two-photon imaging were carried out using Olympus FV1000 with Mai Tai DeepSee Laser (Spectra-Physics) with an excitation wavelength of 900 nm and an emission filter of 515–560 nm. Focused laser injury was induced ain a small area of about 80 μm² within the region of interest at 50 μm below the pial surface using a laser pulse (75% power for 30 s) with a frequency of 8 μs per pixel. Microglia response was recorded every 5 min for a total of 35 min at a depth of 30–70 μm with 2.5-μm z increments and 512 × 512-pixel resolution. In each mouse, 4–6 different sites were recorded. At the end of the experiment, mice were killed by decapitation.