Plan apochromat 0.45 na

Animals were perfused and post-fixed overnight using 4% paraformaldehyde (HistoFix, Roche). Vibratome sections (100-200 mm) were collected in 1x PBS and were incubated in blocking buffer (1x PBS, 0.3% Triton X-100, 10% Donkey serum) at room temperature for 1 hour. Then slices were incubated with primary antibodies in blocking buffer overnight at 4 C. The next day, slices were washed 3 times for 10 min each in 1x PBS with 0.3% Triton X-100 and incubated in secondary antibody solution diluted in blocking buffer overnight at 4 C. We used rabbit anti-GFP (Thermo Fisher, A-11122, 1:500) as a primary antibody to label ChR2-positive cells, anti-mCherry (Novus, NBP2-25158, 1:500) to label hM4D-postive cells. Alexa488 donkey anti-rabbit (Thermo Fisher, A21206, 1:500-1000) and Cy3 donkey anti-chicken (ImmunoJackson, 703-166-155, 1:1000) were used as secondary antibodies. Nuclei were stained with DAPI (Roche, 10236276001, 1:500) together with the secondary antibody solution. Sections were then again washed 3 times for 10 min in 1x PBS with 0.3% Triton X-100 and 1 time in 1x PBS, covered with mounting medium (Dako, C0563) and a glass coverslip. Confocal microscopy was performed on a Zeiss LSM 710 microscope. Images of areas with ChR2-and hMD4-expressing cells, the fiber location and the Neuropixels track labeled with DiD were obtained using a 10x (plan-APOCHROMAT 0.45 NA, Zeiss) objective.