Alexa fluor 488 affinipure donkey anti chicken

Histology was performed on TC slices following intracardial perfusion (4% paraformaldehyde in phosphate-buffered solution (PBS) 0.1 M, pH 7.4). The hemispheres were separated and cut with a 55° angle from the midline according to the rat brain coordinates of Land and Kandler (2002) (link) to preserve pathway from the thalamus to S1 and 60 µm slices were embedded (2% agarose) and cut using a vibratome (Microm HM 650 V, Thermo Fisher Scientific). Slices were rinsed and blocked overnight at 4 °C in 10% normal goat serum (Invitrogen) and 10% normal donkey serum (D9663, Sigma-Aldrich) in PBS containing 0.05% Triton-X and 1% BSA on a shaking Table at 4 °C. Primary antibodies chicken-anti NeuN (1:500; ABN91, Millipore), mouse-anti GAD67 (1:1000; MAb5406, Millipore), and rabbit-anti GABA (1:500; A2052, Sigma-Aldrich) were incubated for 48 h 4 °C to ensure thorough tissue penetration. Secondary antibodies, Alexa Fluor 488-AffiniPure donkey anti-chicken (1:200; 703-545-155, Jackson Immunoresearch), Alexa Fluor 568 goat anti-mouse (1:200; Invitrogen) and Alexa Fluor 647 goat anti-rabbit IgG (1:200; Invitrogen) were incubated at room t° for 3 h. Slices were mounted in Dako fluorescent mounting medium (Dako North America Inc.). Negative control experiments were performed by incubation without primary antibodies.

Colchicine or FG-injected Nts^cre;GFP mice were deeply anesthetized with sodium pentobarbital, transcardially perfused with 10% formalin and brains were post-fixed for 24 h. After dehydration with 30% sucrose the brains were coronally sectioned (30 μm) using a freezing microtome (Leica). Each brain was sectioned into four separate but equally represented series. Immunofluorescence was performed as previously described (Leinninger et al., 2011 (link)). Sections were incubated with chicken anti-GFP (1:2000, Abcam) and mouse anti-tyrosine hydroxylase (TH) (1:1000, Millipore) or rabbit anti-Fluoro-Gold (1:500, Fluoro-Gold chrome) followed by incubation with a 1:200 solution containing species-specific secondary antibodies with fluorescent conjugates (Alexa Fluor^® 488 AffiniPure donkey anti-chicken – Jackson ImmunoResearch, ab13970; donkey anti-rabbit IgG (H + L) Highly Cross Adsorbed Secondary Antibody Alexa Fluor^® 568 – ThermoFisher Scientific, A10042; donkey anti-mouse IgG (H + L) Highly Cross Adsorbed Secondary Antibody, Alexa Fluor^® 568- ThermoFisher Scientific, A10037).