183 Axonal Loss in Narcolepsy Type 1 three times in ultrapure water. Sections were dehydrated in successive solutions for two minutes each: ethanol 70%, 80%, 96%, 100%, 100%, xylene, xylene, xylene. Sections were covered with Entellan® (107961; Merck Millipore, Massachusetts, United States) and coverslips. After deparraffination of the sections, for the SMI312 staining, the antigen was retrieved by washing for five minutes in 10 mM citrate buffer with a pH of 6.0, followed by putting the sections in the citrate buffer in the steam cooker for 30 minutes. They were cooled off to room temperature. After cooling down to room temperature, the sections were washed with TBS for five minutes, blocked with 1% H2O2 in TBS for 30 minutes and washed three times with TBS for five minutes each. Sections were blocked with 3% NGS in TBS for 30 minutes. The primary antibody – Mouse anti human SMI312 (837904; Biolegend, California, United States) was added in a dilution of 1:500 (100 µL incubation + parafilm). Sections were set aside at room temperature for 30 minutes and then incubated overnight at 4°C. The next day, sections were washed three times with TBS for five minutes each time. Then, the secondary antibody – Biotgoat anti mouse (115-065-146; Jackson ImmunoResearch, Cambridge, United Kingdom) – was added in a dilution of 1:400 in TBS for two hours. After washing three times with TBS for five minutes each time, sections were incubated for one hour with avidin-biotin complex (ABC elite kit, PK6100; Vector Laboratories, California, United States) in a dilution of 1:400 with 0.1% Triton x-100 (TBS-T). Sections were washed three times for five minutes, twice in TBS and once in Tris HCL. Staining reaction was accomplished by putting the sections in 3,3-diaminobenzidine (DAB; Sigma, Missouri, United States). Another washing step in Tris HCl followed three times. Dehydration and covering steps were the same as for the Bielschowsky silver staining. The SMI312 staining was used to quantify the percentage of area stained with phosphorylated neurofilament chains (all nine ROIs were assessed). We utilized an in-house developed pixel classifier script (previously described by Frigerio et al. [252], for an example see Supplementary Figure 1) that identified phosphorylated neurofilament medium and heavy chains in axons of all nine ROIs. Myelin stainings For the myelin stainings we used luxol fast blue (LFB; Gurr #16765, Electron Microscopy Sciences, Pennsylvania, United States) and mouse antibovin myelin proteolipid protein antibody (PLP; MCA839G; Bio-Rad Laboratories Inc., California, United States). 6
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