64 Chapter 3 Materials and methods Human Brain Tissue Human brain tissues were obtained from 19MS patients diagnosed by theMcDonald criteria12 and 6 age-matched controlswith non-neurological conditions (Table 1). Controlswere selected from a larger cohort based on the clinical and pathological profile and were excluded if they were recorded as having neurological disorders, cancer or other inflammatory CNS diseases. The rapid autopsy regime of the Netherlands Brain Bank in Amsterdam (coordinator, Dr I. Huitinga) was followed to obtain donor tissues. Participants or next of kin had given informed consent for brain autopsy and for use of their tissue for research purposes. Tissues were fixed in 4% paraformaldehyde, processed, and embedded in paraffin. Tissues were selected and classified based on the size and type of lesion for quantitative analyses. Identification of the lesions was acquired by immunohistochemistry for myelin proteolipid protein (PLP) to detect myelin loss and HLA-DR to detect microglia and macrophage activation13. Table 1. Clinical details of multiple sclerosis and control cases Case Age Sex Diagnosis Duration years PM delay h:min Cause of death Multiple sclerosis 1 50 F SPMS 17 7:35 Euthanasia 2 66 F PPMS 13 9:35 Euthanasia 3 77 F PPMS 24 10:00 Euthanasia 4 54 F Unknown 27 9:25 Respiratory failure 5 63 F Unknown Unknown 10:50 Unknown 6 70 M SPMS 38 9:25 Euthanasia 7 61 M SPMS 18 9:15 Euthanasia 8 60 F SPMS 10 10:40 Euthanasia 9 64 F SPMS 31 10:10 Urinary tract infection by MS 10 56 M SPMS 14 10:10 Unknown 11 71 M Unknown Unknown 7:15 Pneumonia 12 70 M SPMS 38 9:25 Euthanasia 13 45 M PPMS 10 7:45 Pulmonary embolism 14 63 M Unknown Unknown 10:00 Pneumonia / Sepsis 15 63 M Unknown Unknown 11:00 Pneumonia / Sepsis 16 67 M SPMS 38 11:00 Pneumonia 17 77 F Unknown Unknown 9:45 Aspiration pneumonia 18 35 F SPMS 10 10:20 Euthanasia 19 66 F Unknown Unknown 09:30 Euthanasia Controls 1 49 M Control N/A 6:15 Euthanasia 2 56 M Control N/A 14:00 Heart failure 3 81 M Control N/A 5:30 Prostate carcinoma 4 92 F Control N/A 7:00 Pulmonary embolism 5 62 M Control N/A 7:20 Unknown 6 71 M Control N/A 8:55 Pancreatic / rectal carcinoma F = female; M = male; MS = multiple sclerosis; N/A = not applicable; PM = post-mortem; PP = primary progressive; SP = secondary progressive. Immunofluorescence Paraffin sections were immersed in xylene for 5 min for de-paraffinization and rehydrated in descending concentrations of ethanol and washed in phosphate buffered saline (PBS). To reduce autofluorescence sections were incubated in 0.1% glycine for 10 min. Antigen retrieval was performed with citrate or TRIS buffer in the microwave (10 min, 180 Watt). Sections then were incubated with primary antibodies diluted in antibody diluent (Sigma, U3510) overnight, washed and afterwards incubated with Alexa Fluor®-labelled secondary
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