F-J B is used as a marker of cellular degeneration. F-J B histofluorescence was carried out in order to examine neuronal damage/death following TFI. As described previously [12 (link),26 (link)], in brief, the sections were incubated in 0.06% potassium permanganate (KMnO4) (Sigma-Aldrich Co.) for 20 min on an orbital shaker. After washing for two minutes, these sections were reacted in 0.0004% F-J B (Histochem, Jefferson, AR, USA) for 30 min. Thereafter, the brain sections were washed three times for two minutes and placed on a slide warmer until they were fully dried. The slides were cleared using xylene for one minute and coverslipped with dibutylphthalate polystyrene xylene (Sigma-Aldrich Co.).
The count of F-J B positive cells (neurons) was performed to evaluate TFI-induced neuronal death in the CA1 region. In short, according to published methods [4 (link),24 (link)], the image of F-J B positive cells was taken at the magnification of 20X using epifluorescent microscope (Carl Zeiss, Oberkochen, Germany), equipped with a digital camera (DP72) (Olympus), at 450–490 nm of wavelength. F-J B positive cells were taken using image capture software (cellSens Standard) (Olympus). The total number of F-J B positive cells was counted in the same way described above (count of NeuN immunoreactive neurons).
The count of F-J B positive cells (neurons) was performed to evaluate TFI-induced neuronal death in the CA1 region. In short, according to published methods [4 (link),24 (link)], the image of F-J B positive cells was taken at the magnification of 20X using epifluorescent microscope (Carl Zeiss, Oberkochen, Germany), equipped with a digital camera (DP72) (Olympus), at 450–490 nm of wavelength. F-J B positive cells were taken using image capture software (cellSens Standard) (Olympus). The total number of F-J B positive cells was counted in the same way described above (count of NeuN immunoreactive neurons).
