Cy3 conjugated donkey anti rabbit

Following completion of experiments, rats were anesthetized with sodium pentobarbital (100 mg/kg) and transcardially perfused with 0.01 M PBS (phosphate-buffered saline) followed by 10% buffered formalin solution (HT501320, Sigma Aldrich). Brains were removed and stored in formalin with 20% sucrose. All brains were sectioned at 30 μm on a freezing stage microtome (SM2010R, Leica Biosystems). Sections were collected and processed to label for GFP (as an indicator of GCaMP6f or dLight1.2 expression) and/or TH via immunohistochemistry. Antibodies were incubated at 4 °C (washes and other steps at room temperature). Tissues were permeabilized in 0.3% Triton-X 100 for 30 min and blocked in 2% normal donkey serum for 30 min. Sections were incubated in rabbit anti-TH (AB152, Sigma Aldrich) and/or chicken anti-GFP (AB13907, Abcam) antibodies overnight (∼18 h). After KPBS (potassium phosphate-buffered saline) washes (eight changes, 10 min each), secondary antibody (Cy3 conjugated donkey anti-rabbit and AF488 conjugated donkey anti-chicken; Jackson Immunoresearch) was applied and sections were incubated overnight. Sections were then mounted onto glass slides, air dried, and coverslipped with 50% glycerol in KPBS mountant. Only data from subjects with GFP and mCherry expression and correct fiber placement were included in statistical analyses.

For confocal microscopy, rats were perfused with ice-cold saline and 4% paraformaldehyde 3 or 6 weeks after retroviral injections. Brains were processed for immunohistochemistry as previously described [11 (link)]. The following primary and secondary antibodies were used: rabbit polyclonal anti-Iba1 (1:1000, Wako, Japan), chicken polyclonal anti-GFP (1:5000, Abcam, UK), mouse monoclonal anti-gephyrin (1:500, Synaptic Systems, Germany), Cy3-conjugated donkey anti-rabbit (1:200, Jackson Immunoresearch, UK), Cy3-conjugated goat anti-mouse (1:200, Jackson Immunoresearch), biotinylated goat anti-chicken (Vector laboratories), Cy5-conjugated goat anti-rabbit (1:200, Jackson Immunoresearch), Alexa-488 goat anti-chicken (1:200, Invitrogen, Sweden), and Alexa-488 streptavidin (1:200, Invitrogen).