Ds ri2 digital

The sections were observed under a fluorescence microscope (Olympus BX61, Tokyo, Japan). Images were captured using a cooled digital DS-Ri2 digital camera (Nikon, Tokyo, Japan) with NIS-Elements F software. The photomicrographs were taken at an objective magnification factor of ×40 and ×100 to capture the dorsal horn of SC and the area around (and including) the CC. The raw photomicrographs were processed by ImageJ software (National Institutes of Health, Bethesda, MD, USA). First, the fluorescence leakage reduction was performed for the green staining pictures through subtraction of red counter-signal for green fluorescence and then the median filter was used with a radius of 2.0 pixels. The red staining gfap-pictures were exposed only to the median filter with a radius of 5.0 pixels. Subsequently, each picture was adjusted to the threshold method with a triangle thresholding algorithm and analyzed to measure the fluorescence percentage area. Finally, analysis of overlapping regions obtained by dual immunohistochemistry was carried out by merging the green and red staining threshold pictures and calculating the fluorescence percentage area of the overlapping part using Adobe Photoshop (Adobe Inc., San Jose, CA, USA). For the purpose of publication, subtraction of the background was performed and all the figures are slightly contrasted.