Additional Supporting Information may be found in the online version of this article.

GLIA_22276_sm_SuppFig1.tif664KSupporting Information Figure 1. Monoclonal antibody clone 10H9 recognizes breakdown products of NF-L. A) NF-L was digested using cathepsins B and D enzymes and western blotting performed to detect breakdown products with 10H9 mAb. Samples were taken prior to (lane 1) and 10, 20, 40, 60, 120, 180, 240, and 300 min of enzymatic treatment (lanes 2-9 respectively). B) signal intensity (x-axis) of the bands in each lane (molecular weight, y-axis) was analysed using Image J software.
GLIA_22276_sm_SuppFig2.tif712KSupporting Information Figure 2. Quantification of neuronal and myelin proteins in human grey and white matter shows higher concentration of neurofilament compared to myelin. A) Isolated proteins from human grey (lane 1 and 2) and white (lane 3 and 4) matter homogenates were loaded on 10% poly-acrylamide gels (10 μg) and transferred onto nitrocellulose membranes. Amounts of NF-L and MBP were detected. B) signal intensity (x-axis) of NF and MBP (molecular weight, y-axis) were quantified using Image J software. In grey matter the signal of neurofilament is 7.5 times stronger than MBP. In white matter, the signal of NF is 3 times stronger than MBP.
GLIA_22276_sm_SuppTab1.doc28KSupporting Information Table 1. Binding characteristics of NF-L antibodies. Three clones of neurofilament antibodies (4F8.1; 5B4.2 and 10H9) generated in-house and one commercially available neurofilament antibody (DA2) were tested on human tonsil to test specificity. All four antibodies recognised neurofilament antigens in the brain. However, clone 10H9 was the only antibody that did not react with leukocyte antigens in tonsil. All antibodies recognised NF-L breakdown products (not determined for clone 5B4.2).
GLIA_22276_sm_SuppTab2.doc59KSupporting Information Table 2. Number of HLA-DR+ cells containing myelin or axonal proteins. Paraffin sections from MS and control brain tissues were stained for HLA-DR and myelin (PLP or MBP) or axonal (APP, NF-L or non-phosphorylated NF-H) proteins. The number of HLA-DR+ cells containing myelin or axonal antigens was quantified. Additionally, the number of APP+ axonal ovoids per section was determined as a measure of axonal damage. Quantification of total area and number of HLA-DR+ cells was performed using ImageJ software.

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