Goat anti rabbit igg secondary antibody with alexa fluor 594 conjugate

At the described timepoints (3rd, 7th, 14th and 21st days) following surgery, nerves were dissected, rinsed and xed in 4% PFA for 5 h at 4 °C. Then, the sciatic nerves were sectioned using a para n section system. Nerve sections were permeabilized with 0.2% Triton X-100 for another 15 min at room temperature and blocked for 1 h in 10% normal goat serum (1:50, DAKO, USA) at room temperature. The sciatic nerve sections were stained for double immuno uorescence using mouse anti-neuro lament heavy chain antibody (1:500, Santa, sc-32729) and rabbit anti-S100β antibody (1:100, Abcam, Ab52642). Nerve sections were coincubated with primary antibodies overnight at 4 °C, followed by incubation with goat anti-mouse immunoglobulin G (IgG) secondary antibody with Alexa Fluor 488 conjugate (1:100; Abcam) and goat anti-rabbit IgG secondary antibody with Alexa Fluor 594 conjugate (1:100; Abcam) for 2 h at room temperature. Nuclei were stained with DAPI (Ex/Em: 405/430-470 nm). All procedures were accompanied by rinsing three times in PBS for 5 min each. Finally, the slides were photographed with CLSM to obtain the nerve microstructure of the sciatic nerve during regeneration.