Anti gch1 antibody

Cells were fixed in 4% PFA. For immunostaining, brain sections were randomly chosen based on the anatomical location from different serial collecting wells for an individual rat. Each collecting well contained approximately 6–8 serial sections from anterior to posterior at a spacing of every 300 μm. Blocking solution for fixed cells and brain sections included 10% normal donkey serum (Jackson Immunoresearch) in PBS surplus with 0.4% Triton X-100. Primary antibodies (Tyrosine Hydroxylase: millipore; DDC Antibody: Novus Biologicals; Anti-GCH1 Antibody: abcam; Mouse anti-human nuclei monoclonal antibody: Millipore; Ki-67 Monoclonal Antibody: Thermo Fisher Scientific; Anti Iba1: Wako; Anti-CD45: Bioss; Anti-Glial Fibrillary Acidic Protein Antibody: millipore) were diluted in PBS containing 2.5% normal donkey serum and 0.4% Triton X-100 and incubated overnight at 4 °C according to manufacturer recommendations. Secondary antibodies conjugated to Alexa488, Alexa555, or Alexa647 (Molecular Probes) diluted in PBS containing 5% normal donkey serum were incubated for 2 h at room temperature. Nuclear counterstain was visualized with 4′,6-diamidino-2-phenylindole (DAPI, Thermo Fisher). After PBS wash, stained sections were mounted on adhesion microscope glass slides (MXB Biotechnologies).