Alexafluor 594 or 633 goat anti chicken

Immunohistochemistry was performed as described in our previous studies^{6 (link)–8 (link),11 (link)}. In brief, mice were intracardially perfused with 4% paraformaldehyde, and then brains were sectioned (30 mm) and placed in 0.1 M PB until immunohistochemistry. Free-floating sections were washed in 0.1 M PBS for 3 X 10 minutes intervals. Sections were then placed in blocking buffer (0.5% Triton X-100 and 5% natural goat serum in 0.1 M PBS) for 1 hr at room temperature. After blocking buffer, sections were placed in primary antibody (chicken anti-tyrosine hydroxylase,1:2000, Aves Labs, Inc.; rabbit anti-ChR2, 1:500, American Research Products) overnight at room temperature. After 3 X 10-minute 0.1 M PBS washes, sections were incubated in secondary antibody (AlexaFluor 488 goat anti-rabbit; AlexaFluor 594 or 633 goat anti-chicken, Life Technologies) for 2 hours at room temperature, followed by subsequent washes (3 X 10 minute in 0.1 M PBS). Later, sections were incubated in NeuroTrace (435/455 blue fluorescent Nissl stain, ThermoFisher Scientific) for 1 hour, followed by 3 X 10 minute 0.1 MPBS then 3 X 10-minute 0.1 M PB washes. After immunostaining, sections were mounted and coverslipped with Vectashield HardSet mounting medium (Vector Laboratories) and imaged on a Leica DM4 P epifluorescence microscope.

Immunohistochemistry was performed as previously described by (Al-Hasani et al., 2013; Kim et al., 2013; McCall et al., 2015 (link)). In brief, mice were intracardially perfused with 4% paraformaldehyde, and then brains were sectioned (30μm) and placed in 0.1 M PB until immunohistochemistry. Free-floating sections were washed in 0.1 M PBS for 3 × 10 minutes intervals. Sections were then placed in blocking buffer (0.5% Triton X-100 and 5% natural goat serum in 0.1 M PBS) for 1 hr at room temperature. After blocking buffer, sections were placed in primary antibody (chicken anti-tyrosine hydroxylase,1:2000, Aves Labs, Inc.; rabbit anti-nociceptin, 1:500, abcam) overnight at room temperature. After 3 × 10-minute 0.1 M PBS washes, sections were incubated in secondary antibody (AlexaFluor 488 goat anti-rabbit; AlexaFluor 594 or 633 goat anti-chicken, Life Technologies) for 2 hours at room temperature, followed by subsequent washes (3 X 10 minute in 0.1 M PBS). Later, sections were incubated in NeuroTrace (435/455 blue fluorescent Nissl stain, ThermoFisher Scientific) for 1 hour, followed by 3 × 10 minute 0.1 MPBS then 3 × 10-minute 0.1 M PB washes. After immunostaining, sections were mounted and coverslipped with Vectashield HardSet mounting medium (Vector Laboratories) and imaged on a Leica TCS SPE confocal microscope.