Tcs sp8 fluorescence laser scanning confocal microscope

Briefly, 40-μm-thick coronal slices were obtained and the procedures for immunohistochemistry were performed similar to what we did before (Wei et al., 2015 (link)).
To determine the input patterns of the mOT, every sixth sections of the RV-labeled brain slices were collected and stained with DAPI. Four coronal slices from the VTA (AP from about −3.00 mm to −3.80 mm, spaced 240 µm from each other) per mouse were immunohistochemically stained for the tyrosine hydroxylase (TH, Abcam, Lot#: 2552365).
To determine the specificity of ChR2-mCherry expression in the VTA of DAT-Cre mice, 4 coronal slices from the VTA (AP from about −3.00 mm to −3.80 mm, spaced 240 µm from each other) per mouse (n = 3) were collected and immunohistochemically stained for the TH.
To detect the c-fos expression evoked by optical-manipulation, mice were put in an open field and received laser illumination for 10 min and were sacrificed 1.5 hr later. Every sixth brain sections (AP from about 1.40 mm to 0.50 mm) from three mice were collected and immunohistochemically stained for c-fos (Cell Signaling, #2250).
All the images were then captured and analyzed with TCS SP8 fluorescence laser scanning confocal microscope (Leica) and ImageJ software.

The mice were anesthetized with chloral hydrate (10% W/V, 500 mg/kg body weight, i.p.), and perfused transcardially with PBS (5 min), followed by ice-cold 4% paraformaldehyde (PFA, 158127 MSDS, sigma) dissolved in PBS (5 min). The brain tissues were carefully removed and post-fixed in PBS containing 4% PFA at 4°C overnight, and then equilibrated in PBS containing 25% sucrose at 4°C for 72 h. The 40 μm thick coronal slices of the whole brain were obtained using the cryostat microtome and stored at -20°C.
For RV or AAV labeled samples, every sixth section of the brain slices were selected, stained with DAPI, washed with PBS, mounted with 90% glycerol (in PBS) and sealed with nail polish.
For PRV-152 labeled samples, the procedures for immunohistochemistry were performed as before (Wei et al., 2015 (link)). Every sixth section of the brain slices were selected and stained with GFP (abcam, ab290, 1: 1000) and DAPI, and then mounted and sealed as described above.
All of the images were captured with the TCS SP8 fluorescence laser scanning confocal microscope (Leica, China) or the Olympus VS120 virtual microscopy slide scanning system (Olympus, Shanghai, China).