Cy 3 affinipure donkey anti goat igg

Brainstems of mice were collected after 4% PFA perfusion and post-fixed for 4 hours on ice. After cryoprotected in 30% (w/v) sucrose for two nights at 4 °C, brainstem was immerged into OCT and sectioned at 50 μm using the cryostat for free-floating section staining. Sectioned tissues were blocked with 10% goat serum in PBST (PBS containing 0.1% Triton X-100) for 1 hour and incubated with primary antibodies at 4 °C overnight. After washing with PBST, secondary antibodies were applied for 2 hours at room temperature. Fluorescence images were taken using Nikon C2 confocal system. Primary antibodies including: rabbit anti-GFP (Invitrogen, A11122, Lot# 1925070; 1:1000), rabbit anti-c-Fos (Santa Cruz Biotechnology, sc-52, Lot# B0112; 1:1000), mouse anti-NeuN (MilliporeSigma, MAB377, Lot#3205920; 1:1000), goat anti-WGA (Vector Laboratories, AS-2024, Lot#T1112; 1:1000), guinea pig anti-Synaptophysin 1 (Synaptic System, 101 004; 1:200). Secondary antibodies including: goat anti-rabbit IgG-Alexa Fluor-488 (Invitrogen, A11008, Lot # 1797971; 1:500), goat anti-rabbit IgG-Alexa Fluor-555 (Invitrogen, A21429, Lot # 1683674; 1:500), Goat anti-mouse IgG-Alexa Fluor-Cyanine5 (Invitrogen, A10524, 1:500), Cy™3 AffiniPure Donkey Anti-Goat IgG (Jackson ImmunoResearch, 705165147, Lot # 148575; 1:500), goat anti-guinea pig IgG-Alexa Fluor-555 (Invitrogen, A21435; 1:500).