40 Chapter 2 Materials and methods Human brain tissue Human brain tissue was obtained at autopsy from 42 people with multiple sclerosis and 12 age-matched cases with no neurological disorders or peripheral inflammation. Biopsy material from MRI tumour-like lesions was taken from 4 patients with suspected multiple sclerosis. Patient data and clinical details are summarized in Table 1. The rapid autopsy regimen of the Netherlands Brain Bank in Amsterdam (coordinator Dr. I. Huitinga) was used to acquire the samples, with the approval of the Medical Ethical Committee of the Amsterdam UMC. All participants or next of kin had given informed consent for autopsy and use of their tissues for research purposes. Tissue samples from multiple sclerosis cases were selected from regions of interest after ex vivo MRI57,58. Brain samples were cut in half and fixed in 10% formalin and embedded in paraffin or snap-frozen and stored in liquid nitrogen. The cases and lesions were selected from a large cohort of multiple sclerosis cases based on the size and lesion type for quantitative analysis. Lesion stages were based on immunohistochemical detection for myelin proteolipid protein (PLP) to detect areas of myelin loss and expression of human leukocyte antigen DR (HLA-DR)59,60. Briefly, active lesions were characterised by a focal area of myelin loss filled with myelin-laden ‘foamy’ macrophages; chronic active lesions were identified by a rim of activated microglia/macrophages surrounding a hypocellular centre, and inactive white matter lesions as a demyelinated area with few or no HLA-DR+ cells. The normal appearing white and grey matter in the same tissue blocks from multiple sclerosis cases and control white and grey matter from age-matched controls were analysed as reference samples for the expression of cell markers. Immunohistochemistry Paraffin sections were de-paraffinized using xylene, rehydrated through descending alcohol solutions and washed in phosphate buffered saline. Frozen sections were air-dried and fixed in 2% paraformaldehyde for 10 mins at room temperature and washed in phosphate buffered saline. Endogenous peroxidase activity was blocked using 0.3% (w/v) H2O2. Following washing, paraffin sections underwent antigen retrieval in citrate or TRIS/EDTA buffer in a water bath at 95°C for 30 mins. After cooling, sections were washed and incubated in primary antibodies overnight in antibody diluent (Immunologic, Duiven, TheNetherlands). After washing, sections were incubated in the appropriate secondary antibodies. Horseradish peroxidase labelled secondary antibodies were developed using 3,3’-diaminobenzidine (1:50, DAB, DAKO, CA) for 10 min after which sections were washed in tris buffered saline. Liquid permanent red (1:100, DAKO) was used to visualise alkaline phosphatase labelled antibodies. Sections were washed with tris buffered saline, counterstained with haematoxylin, washed and mounted in Aquatex (Merck, Darmstadt, Germany). For immunofluorescence, sections were incubated with Alexa Fluor labelled secondary antibodies in antibody diluent for 90 mins and nuclei were stained with 4’,6-diamidino-2-phenylindole. Appropriate negative controls were used by omitting the primary antibodies.
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