Immunohistochemical Analysis of TG and spinal trigeminal nucleus caudalis

Immunohistochemical staining of TG and spinal trigeminal nucleus caudalis was done following our previously published work [13 (link)]. Briefly, the sections from TG were stained with primary antibody cleav Casp3 and secondary antibody (Table 3). The spinal trigeminal nucleus caudalis sections were stained with primary antibodies C-Fos and GFAP followed by secondary antibodies (Table 3). Nuclear staining was done using 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI, 1:100, Nacalai Tesque, Inc., Kyoto, Japan). Isotypes (goat IgG bs-0294P, and rabbit IgG bs-0295P, Bioss Antibodies, Boston, MA, USA) and only secondary antibodies were used as positive and negative control. Images were observed and acquired by confocal laser-scanning microscope (LSM 700, Carl Zeiss, Oberkochen, Germany).