Molecular neuroscienceDifferential, strain-specific cellular and subcellular distribution of multidrug transporters in murine choroid plexus and blood–brain barrier
Section snippets
Animals
Adult mice were purchased from commercial suppliers: C57BL/6 (Jackson Laboratories, Bar Harbor, ME, USA), SVJ (Institute for Laboratory Animal Sciences, University of Zurich), Swiss (Harlan, Horst, The Netherlands), and FVB wild type, Mdr1a0/0, Mrp10/0 inbred strains (Taconic Farms, Hallingore, Denmark). Generation and characterization of Mrp20/0 mice will be described elsewhere (Vlaming et al., in preparation). All mice were housed in a 12-h light/dark cycle with free access to food and water.
Characterization of antibodies
The distribution of ABC-transporters in the BBB and choroid plexus was analyzed in sections prepared from fresh frozen tissue and weakly fixed by immersion in a low concentration of paraformaldehyde. This procedure was selected because pilot experiments in perfusion-fixed tissue resulted in a low signal-to-noise ratio and unspecific staining of numerous organelles (not shown).
At first, commercially available monoclonal antibodies raised against human-specific sequences were tested. Good results
Discussion
Numerous studies have investigated the expression and distribution of ABC-transporters at the BBB and in the choroid plexus, with partially controversial findings that might reflect discrepancies in mRNA and protein expression, species specificity, and technical differences. Our results show that a strong signal-to-noise ratio can be achieved for immunofluorescence staining in weakly fixed tissue, a prerequisite for high resolution imaging using laser scanning microscopy. Using this approach,
Acknowledgments
We are grateful to Corinne Sidler and Franziska Parpan for outstanding technical assistance. This study was supported by the Swiss National Science Foundation (National Center of Competence in Research “Neural plasticity and repair”) and the Dutch Cancer Society, grant NKI 2003–2940.
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