Guinea pig anti vglut2

Anterograde tract tracing from the Vsp to the PBN regions was performed using BDA (D1956, Invitrogen) in 15 rats. In each rat, 0.2 μl of 2% (w/v) BDA in distilled water was injected into the Vo (5 rats), Vi (5 rats) or Vc (5 rats). After a period of 5 days, the rats injected with BDA were sacrificed. Similar to the procedure of the FG and TMR injection experiments, the brainstem of the rats was cut into transverse sections in series, and one series of sections through Vo, Vi or Vc was directly incubated with Fluorescein Avidin D to detect the location and extent of the BDA injection site.
The sections through the PBN from the rats that had a proper BDA injection site were subsequently incubated overnight at room temperature with a mixture of 1 μg/ml mouse anti-NeuN antibody (MAB377; Millipore, Billerica, MA) and 2 μg/ml guinea pig anti-VGLUT2 (135,404, Synaptic Systems, Goettingen, German) in PBS that contained PBS-XCD, followed by 6 h at RT with a mixture of 5 μg/ml Fluorescein Avidin D (Vector), 5 μg/ml Alexa 594-conjugated goat anti-guinea pig IgG antibody (A-11076, Invitrogen) and 5 μg/ml Alexa 647-conjugated donkey anti-mouse IgG antibody (A-31571, Invitrogen).