Additional file 1: Table S1. of Complement is activated in progressive multiple sclerosis cortical grey matter lesions
Creators: name: Watkins, Lewis
name: Neal, James
name: Loveless, Sam
name: Michailidou, Iliana
name: Ramaglia, Valeria
name: Rees, Mark
name: Reynolds, Richard
name: Robertson, Neil
name: B. Morgan
name: Howell, Owain
Publisher: name: Figshare
Year: 2016
Subjects: subject: Biochemistry
subject: Medicine
subject: Microbiology
subject: FOS: Biological sciences
subject: Cell Biology
subject: Molecular Biology
subject: Neuroscience
subject: Physiology
subject: Immunology
subject: FOS: Clinical medicine
subject: Developmental Biology
subject: Cancer
subject: Inorganic Chemistry
subject: FOS: Chemical sciences

description: Demographic and pathological details for cases used in this study. All cases were retrospectively confirmed as MS, non-inflammatory or inflammatory (viral encephalitis or ischaemia) following detailed analysis of patient health records and full neuropathological work up. Region matched sections (FFPE or cryosections) were available for all cases from the frontal cortex (sup. Frontal gyrus), cingulate cortex and hippocampus and temporal (inf.) gyrus. Tissue sections from inflammatory control cases were selected based on region-matching and not on the presence of pathology in the particular block. Examples of myelin and inflammatory cell staining are shown in Additional file: Figure S1. Abbreviations:  PMD- post-mortem delay from death to tissue processing (hrs); Disease activity- neuropathologically defined disease activity based on the presence (Active) or absence (Stable) of active inflammatory lesions; Disease duration is calculated from retrospectively determined time of disease onset; Tissue- available formalin-fixed paraffin embedded (FFPE) or cryopreserved tissue blocks; Quantified lesions- number of GM or WM lesion regions of interest analysed in this quantitative study. Table S2. Primary antibodies used in this study, retrieval conditions and commercial source. Citrate- 100mM sodium citrate, 10mM citric acid, pH 6.0; TRIS/ EDTA- 10mM Tris, 1mM EDTA, pH 8.5. Table S3. Complement cell-associated expression was not related to age of death, post-mortem delay or whether death was infection (inflammatory) or non-infection related. Linear regression and Pearson’s correlation analysis (Age of death and PMD (post mortem delay) versus mean C3b+ cell count per case) or Mann-Whitney test of mean C3b+ cell count between those cases with an inflammatory-related or non-inflammatory disease noted at death. Figure S1. Non-neurological controls and inflammatory neurological controls used in this study. Control cortical grey and subcortical white matter was characterised by a uniform pattern of anti-MOG staining (A- D) and small numbers of ramified HLA-D+ microglia (E- H). Inflammatory controls were selected on the basis of available region-matched blocks and frequently displayed an unremarkable pathology. Occasional areas of microglial activation were noted in some sections of cortical and subcortical tissue (I- P; ischaemic encephalitis inflammatory control), whilst perivascular cuffs of activated microglia and macrophages were sometimes seen in cases of viral encephalitis (Q- X). Scale bars= W (and all low power images) 100µm; X (and all high power images) 20µm. Figure S2. Complement recognition fragment, activation products and regulator expression in MS, non-MS inflammatory controls (IC) and non-neurological control (Ctrl) grey matter. 400x magnification images of C1q (A-C), C1-inhibitor (D-F), Bb (G-I), factor H (J-L), C3b-iC3b (M-O), MAC (P, Q) and clusterin (R, S) in deeper cortical laminae of control (Ctrl; left column &amp; P, R), MS (central column &amp; Q, S) and non-MS inflammatory controls (right column only) showing the cell-specific and often robust pattern of anti-complement immunostaining seen in MS (and to a lesser extent, non-MS inflammatory controls) cortical grey matter in comparison to non-diseased controls. Scale bar= 20µm. Figure S3. Complement recognition fragment, activation product and regulator expression in subcortical white matter samples. 400x magnification images of C1q (A-C), C1-inhibitor (D-F), Bb (G-I), factor H (J-L), C3b-iC3b (M-O), MAC (P, Q) and clusterin (R, S) in sub-cortical white matter of non-neurological control (Ctrl), MS and non-MS inflammatory controls (IC), showing the cell-specific and often robust pattern of anti-complement immunostaining seen in MS white matter in comparison to non-neurological controls. Scale bar= 20µm. Figure S4. Examples of complement immunolabelling in Alzheimer’s disease (AD). Two AD cases (age of death, 85 and 96yrs; gender, both female; post-mortem delay, 30 and 19hrs, respectively), showed complement specific immunolabelling with anti-C1q, C3b-iC3b and C9neo of cells resembling neurons and glia (similar to that seen in MS and controls), as well as diffuse labelling of amyloid plaques (for anti-C1q, C3b and C9neo antibodies). Cellular immunolabelling of C1INH and clusterin resembles that seen in control, MS and inflammatory control cases. C5aR+ glia were also seen in the AD deep grey matter. All images captured at 200x magnification. Figure S5. Complement activation in cortical grey matter is not related to underlying white matter inflammation. Grey matter lesions of the deeper cortical laminae are characterised as type I (involving both the white and grey matter) or type IV (involving the entire vertical extent of the cortex but without affecting the underlying white matter). Our analysis revealed there to be no difference between the density of C1q (A,B and D) and C3b-labelled cells (C) in type I grey matter lesions (that are associated with white matter lesions) and type IV lesions. Scatter graphs of type I versus type IV cell-specific counts for C1q and C3b quantitative immunostaining compared by Mann-Whitney test. (PDF 1849 kb)