Tyramide Signal Amplification for Immunohistochemical Detection of Tyrosine Hydroxylase in Mouse Brain

After receiving an intraperitoneal injection of a lethal dose of pentobarbital (Sigma-Aldrich, St. Louis, MO, USA), mice (n = 5) were transcardially perfused with cold PBS, and followed by cold 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.2). Frozen sections with 16-μm thickness were processed for the IHC with the tyramide signal amplification (TSA) technique in a free-floating manner, as in our previous reports (Okita et al., 2012 (link); Morigaki and Goto, 2015 (link)). Briefly, rabbit polyclonal antibody against TH (1:100,000) was used as a primary antibody. To detect the bound antibody, we used the Histofine Simple Stain Kit (Nichirei, Tokyo, Japan) and the TSA Plus Cyanine 3 System (Perkin Elmer, Shelton, CT, USA).