Alexa fluor 647 conjugated donkey anti chicken

Sections were blocked in 1x PBS - 0.3% Triton containing 3% donkey serum (Jackson ImmunoResearch, West Grove, USA), for 1 h at room temperature followed by incubation in primary antibody solution: chicken anti-TH (1:1000; Millipore, USA) or rabbit anti c-Fos (1:500; Santa Cruz Biotechnology, Dallas, TX, USA) in 1x PBS - 0.1% Triton containing 3% donkey serum for 48 h at 4 °C. Sections were then washed 4 times (10 min each) with 1x PBS and immediately transferred to secondary antibody solution: AlexaFluor 647-conjugated donkey anti-chicken (1:1000; Jackson ImmunoResearch, West Grove PA, USA) or Cy3 donkey anti-rabbit (1:500, Jackson ImmunoResearch, West Grove, PA, USA) and containing a DNA-specific fluorescent probe (DAPI; 1:50,000) in 1x PBS containing 3% donkey serum for 2 h at room temperature. Sections not processed for immunohistochemistry were incubated in 1x PBS - 0.3% Triton containing 3% normal donkey serum (Jackson ImmunoResearch, West Grove, USA) and DAPI (1:50,000) for 1 hr. Sections were washed 4 times (10 min each) in 1x PBS and mounted onto glass slides. Slices were allowed to dry and were coverslipped using polyvinyl alcohol (PVA) mounting medium with DABCO (Sigma, MO, USA). Stereotaxic coordinates were determined using brain atlases for rat⁶¹ and mouse⁶² .