Streptavidin conjugated hrp

Slices were incubated in 3% triton X-100 (Solarbio, Beijing, China) 0.01 M PBS for 4 minutes, and were blocked with 10% sheep serum for 15 minutes at room temperature and then incubated with 1° antibody (1:1000 Anti -Tyrosine Hydroxylase AB152, Millipore, USA) at 4 °C overnight. Slices were rinsed with 0.01 M PBS (Solarbio, Beijing, China) and then incubated in biotin conjugated 2° antibody (1:150, Maixin, Fuzhou, China) for 30 minutes, and then incubated in streptavidin conjugated HRP (1:150, Maixin, Fuzhou, China) for 15 minutes. DAB (DAB-1031 Kit, Maixin, Fuzhou, China) was used for coloration of the slices. Slices were dehydrated with an alcohol gradient and made transparent with TO. Neutral balsam was used to close the slides and digital pictures were collected on a microscope, as described above.

Briefly, 10% goat serum (Maixin, Fuzhou, China) was used as the serum blocker for 15 minutes at room temperature. Then anti-tyrosine hydroxylase primary antibody (AB152, Millipore, USA, diluted 1:1000 with 0.01M PBS) was applied at 4°C overnight. Slides were rinsed with 0.01 M PBS-0.1% Triton X-100 (Solarbio, Beijing, China) and were incubated in biotin conjugated secondary antibody (1:150, Maixin, Fuzhou, China) for 30 minutes, and then incubated in streptavidin conjugated HRP (1:150, Maixin, Fuzhou, China) for 15 minutes at room temperature. DAB (DAB-1031Kit 20×, Maixin, Fuzhou, China) was used as the chromogen. Then the slides were rinsed with TO (xylene substitute, Cengxi, Guangxi China), gradient ethanol and distilled water. Then they were stained in Cresyl Violet acetate (C5042-10G, Sigma, USA) plus anhydrous sodium acetate and acetic acid, namely Nissl stain, for 20 minutes at room temperature, followed by sequential rinsing in distilled water, gradient ethanol (70%, 80%, 95% alcohol, 1 minute each) and a differentiation solution (ethanol: chloroform: diethyl ether = 1:1:1) for 5 minutes. Finally, the slides were dehydrated with ethanol, cleared with TO and closed with neutral balsam.