Human control brain tissue specimens, both frozen and paraffin-embedded, consisting of neocortex and white matter, were obtained at autopsy from patients without neurological disease and neuropathologic abnormalities. Additionally, frozen brain tissue was obtained from a multiple sclerosis patient to study gliotic white matter in which MLC1 has a normal localization but MLC1 expression is increased [
3]. During life, all patients or their next of kin had given consent for autopsy and the use of brain tissue for research purposes.
Human glioblastoma multiforme tissue (frozen) and brain biopsy tissue from MLC patients (both frozen and paraffin-embedded) were obtained for diagnostic purposes and used for research purposes with informed consent of patients and families. Light-microscopy of glioblastoma tissue was used to select areas that display characteristic glioblastoma features, including necrosis, giant cells and endothelial proliferation, for further studies. Brain biopsies were obtained in the following MLC patients: EL18 (decreased MLC1 expression [
2], frozen tissue), EL649 (homozygous for the c.733G > C (p.Ala245Pro) mutation; paraffin-embedded tissue), and EL746 [compound-heterozygous for the c.268-1G > A and c.597 + 1G > A mutations, leading to splice-defects and truncation of the protein (p.Cys90-Val107del and p.Val200-Ala238del); paraffin-embedded tissue].
Immunohistochemistry was performed as described previously [
7] with antibodies against the following proteins: MLC1 (polyclonal; 1:200 [
3]); merosin (monoclonal; 1:500 [
11]); aquaporin-4 (polyclonal; 1:200, Chemicon, Huissen, The Netherlands); Kir4.1 (polyclonal; 1:200, USBiological, Swampscott, Massachusetts), β-dystroglycan (monoclonal; 1:100, Santa Cruz, Heidelberg, Germany), α-dystroglycan (monoclonal; 1:100, UpstateCellSignaling solutions), agrin (monoclonal; 1:2500 [
26]); glial fibrillary acidic protein (GFAP, polyclonal; 1:500, DAKO, Heverlee, Belgium) and NeuN (monoclonal; 1:1600, MAB377, Chemicon). In all slides, except the slides used for double stainings, the nuclei are counterstained with Haematoxylin. Double immunofluorescence staining was performed as described [
3] with agrin, β-dystroglycan (monoclonal; 1:100) or syntrophin (monoclonal; 1:100, Affinity BioReagents, Raamdonksveer, The Netherlands) versus MLC1. In negative controls the primary antibodies were omitted. Double-stainings were performed as described previously [
25]. In frozen tissue double-stainings were done with agrin (red) antibodies versus AB4 (blue, polyclonal; 1:200, Lab Vision–NeoMarkers, Fremont, CA, USA) antibodies and in paraffin tissue with agrin or α-dystroglycan antibodies versus NeuN or GFAP. For the agrin (brown)/NeuN (blue) double-stainings, agrin was visualized with biotinylated secondary antibodies (DAKO) and NeuN with poly-HRP (Immunologic, Duiven, The Netherlands). For the agrin (red)/GFAP (blue) double-stainings, agrin was detected with poly-HRP (Immunologic) and GFAP with biotinylated secondary antibodies (DAKO). In negative controls the primary antibodies were omitted.