Alexa fluor 647 conjugated affinipure donkey anti guinea pig

Mice were anesthetized with an i.p. injection of Tribromoethanol (250 mg/Kg body weight) and transcardially perfused with ice-cold 0.1 M phosphate buffer (PB) (pH 7.4). Brains were removed and rapidly frozen in OCT using dry ice and 100% ethanol. 5 μm-thick coronal sections of the brainstem were obtained using a cryostat, thaw mounted on microscope slides (Fisherbrand Superfros Plus, Fisher Scientific) and air-dried for 15 min. Sections were fixed by immersion in ice-cold 95% ethanol at 4°C for 30 min followed by acetone for 1 min at RT. Prior to the staining, sections were washed in 0.1 M PB and incubated 20 min at RT in 0.1 M PB blocking solution containing 2% normal goat serum (NGS) and 0.2% Triton X-100. Afterward, sections were incubated at 4°C overnight with the primary antibodies diluted in the same blocking solution (1:100, Rabbit anti-CAST, and 1:100 Rabbit anti-ELKS, T99; 1:100, polyclonal Guinea pig anti-Vglut1, Synaptic Systems). Subsequently, sections were rinsed 3×10 min in 0.1 M PB and incubated with the secondary antibodies diluted in 0.1 M PB containing 0.1% Triton X-100 for 2h at RT 1:200 (Cy 2 AffiniPure goat anti-rabbit IgG (H+L), Jackson Immunoresearch; 1:200, Alexa Fluor® 647-conjugated AffiniPure donkey anti-guinea pig, Jackson Immunoresearch). After 3×10 min in 0.1 M PB washed sections were mounted using Fluoromount-G® mounting medium (SouthernBiotech).