182 Chapter 6 Appendix A – Supplementary methods Tissue processing Brains were removed from the skull fixed in 10% buffered formalin for four weeks. Tissue blocks were extracted from the previously mentioned regions (from the right hemisphere in case of cortical regions) and subsequently embedded in paraffin in preparation for microtomy. Paraffin blocks were cut in 20 µm thick sections for all regions except for the midbrain, which had already been cut in 6 µm thick sections prior to the onset of this project. Sections were mounted on glass slides (SuperFrost PlusTM, Thermo Fisher Scientific, Massachusetts, United States) and dried vertically overnight at room temperature, then horizontally for three hours at 45°C. After deparaffinization in xylene, the sections were rehydrated in graded series of ethanol and then rinsed in demi water. The Bielschowsky silver staining was performed similar to Preziosa et al. [239], the SMI312 staining similar to Luchicchi et al. [243], and myelin stainings similar to Huitema et al. [272]. Axonal stainings The sections were stained for axons with Bielschowsky silver staining, or with SMI312 (837904, Biolegend, California, United States), a mixture of monoclonal antibodies directed against phosphorylated NfM and NfH. For the Bielschowsky staining, after deparraffinization, the sections were washed in ultrapure water. Then, 75 ml of ammonium hydroxide was put open in the fume hood to evaporate. The sections were put in 200 ml of a 20% silver nitrate solution for 20 minutes in the dark and afterwards washed with ultrapure water three times for five minutes. The evaporated ammonium hydroxide was stirred into the previously used 20% silver nitrate solution, until there was no more brown precipitation. At that point, four more drops of ammonia were added. The sections were put in this solution for twenty minutes in the dark, after which they were washed with ultrapure water and ammonia (eight drops of ammonia in 200 ml ultrapure water). Fifteen drops of developer (mixture of 40 mL formaldehyde 37%, 200 mL Milli Q® (Merck, New Jersey, United States), two drops of concentrated nitric acid and 1 gram of citric acid) were added to the silver nitrate and ammonia solution and the solution was stirred well. The sections were put in this solution for approximately 10 minutes. The result was microscopically checked, and the sections were taken out of the solution at the point that axons coloured black and there was also some background colouring. Sections were subsequently washed in seven steps of five minutes: three times in ultrapure water, once in Hypo (5% sodium thiosulfate) and again
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