Golgi–Cox staining was performed using an FD Rapid GolgiStain Kit (FD Neurotechnologies) according to the manual and as previously described [45 (link)]. Following impregnation steps, the tissues were shock-frozen in iso-pentane solution precooled in dry ice and afterwards cut with cryostat in sections with 90 μm thickness and mounted in gelatin-coated glass slides. The cut tissues were allowed to dry for at least overnight, then stained by immersing them in a mixture of staining solution from the kit, dehydrated in ethanol, cleared in xylene, and mounted using Eukitt (O. Kindle) mounting media.
For analysis purposes, only impregnated neurons with diameter of at least 20 μm that were located within the laminae II to V of the spinal dorsal horn were chosen. Moreover, only secondary or tertiary dendrites that were projected in the direction of the dorsal horn were further analyzed. Images were captured using an upright microscope (Nikon NiE, Nikon) equipped with Nikon Plan Apo objective set and high resolution CCD camera (Nikon DS-Ri1, Nikon). Acquisition process was driven by NIS-Elements software 4.1 (Nikon).
For analysis purposes, only impregnated neurons with diameter of at least 20 μm that were located within the laminae II to V of the spinal dorsal horn were chosen. Moreover, only secondary or tertiary dendrites that were projected in the direction of the dorsal horn were further analyzed. Images were captured using an upright microscope (Nikon NiE, Nikon) equipped with Nikon Plan Apo objective set and high resolution CCD camera (Nikon DS-Ri1, Nikon). Acquisition process was driven by NIS-Elements software 4.1 (Nikon).
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