Ab117112

The slice preparations were followed by previously described. One slice from each segment was stained with Masson’s trichrome to locate the ablation sites. Tyrosine hydroxylase (TH), neuronal nitric oxide synthase (nNOS) and calcitonin gene-related peptide (CGRP) were used to detect the efferent sympathetic, parasympathetic and afferent sensory nerves.
After dewaxing, gradient hydrating, antigen retrieval and endogenous peroxidase removal, the serial slices were homologous serum (AR1009, BOSTER, used at 10%; SL050, Solarbio, Beijing, China, used at 10%) blocked and then incubated overnight at 4 °C with TH polyclonal antibody (AB117112, Abcam, used at 1:500), monoclonal anti-CGRP (C7113, Sigma, used at 1:200) and nNOS (AB1376, Abcam, used at 1:200), respectively; then, they were incubated with the corresponding secondary antibodies (PV-6000, PV-9000, Origene; A0181, Beyotime) at 37 °C for 30 min. DAB solution (G1212, Servicebio Technology, Wuhan, China) and hematoxylin dye (G1004, Servicebio Technology, Wuhan, China) were used successively for chromogenic reaction and staining. A fluorescence microscope (OLYMPUS, BX53, Japan) was used for imaging. Absent or low expression indicated that the nerves were completely or partially destroyed.

To investigate whether hydrocephalus altered the localization of TH in the striatum, male rats, 3 per group, were deeply anesthetized as described above and perfused transcardially with 4% paraformaldehyde in 0.1 M PB. The striatum was cryosectioned into 25-μm-thick coronal sections. Sections of interests were first treated with 1% H₂O₂ and 0.4% Triton X-100 in 0.1 M PB for 30 min. The sections were subsequently immersed in 10% normal goat serum and 0.4% Triton X-100 in 0.1 M PBS for 1 h, then incubated with rabbit anti-TH antibody (ab117112; Abcam) in 0.1 M PBS overnight at 4 °C. After several rinses in PBS, the sections were incubated with the biotinylated goat anti-rabbit secondary antibody (AP132B; Millipore), and then the standard avidin–biotin-horseradish peroxidase reagents (PK-6100; Vector Laboratories). Sections were then reacted with 0.05% DAB and 0.01% H₂O₂ in 0.05 M TB. Reacted sections were mounted on slides, dehydrated, and cover-slipped.