184 Chapter 6 After deparraffination, the LFB tissue sections were incubated in a 0.1% LFB solution in 96% ethanol in a stove at 59 °C overnight. All sections were washed and differentiated one by one. Washing steps were performed consecutively in 96% ethanol (2–3 seconds) and ultrapure water (3 seconds). Immediately thereafter, differentiation steps were performed in 0.05% Lithium carbonate solution (554-13-2; Merck Millipore, Massachusetts, United States) for five seconds and 70% ethanol (5–7 seconds) until decolouring of the cortical GM, whilst WM remained blue. The latter step was performed carefully and was checked microscopically to ensure homogeneity of the LFB stainings. The differentiation steps were repeated if needed. The sections were then rinsed in ultrapure water. Finally, the samples were dehydrated in a series of ethanol 96% (3–5 minutes), 100% (2 × 5 minutes) and xylene (3 × 5 minutes). Dehydration and covering steps were the same as for the axonal stainings. PLP staining was performed similar to the SMI312 staining and the differences are hereafter listed. The antigen retrieval steps were done in 10 mM Tris-EDTA (pH 9.0). Aspecific staining was blocked with 1% BSA in TBS-T for 30 minutes at room temperature. The primary mouse anti-PLP monoclonal antibody (MCA839G; Bio-Rad Laboratories Inc., California, United States) was diluted 1:500 in the blocking solution overnight in a humid chamber at 4 °C. Incubation with the secondary antibody was performed after rinsing the next day, using biotin Donkey anti-mouse IgG antibody (715-065-151; Jackson ImmunoResearch, Cambridge, United Kingdom) diluted in 1:400 blocking solution for two hours at room temperature. All stained samples were photographed and digitalized for analysis using a Vectra Polaris Automated Quantitative Pathology Imaging System (Akoya Biosciences, Massachusetts, United States).
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