Novel PMCA2 Mutation and Hearing Loss in Mouse Figure 6. Activity of recombinant PMCA2 pumps in CHO cells. Cells were transiently co-transfected with the PMCA2 variants and cytAEQ, or only with cytAEQ (control). They were then perfused with KRB supplemented with 1 mM CaCl2. 100 mM ATP was used to produce a transient Ca2+ increase (A). The histograms in (B) and (C) show the means6SD of Ca2+ peaks and the half peak decay times, respectively (n indicates the number of experiments considered for the statistical analysis), * p,0.01 calculated with respect to the wt pump. doi:10.1371/journal.pgen.1000238.g006 structural analysis was carried out on this genetic background. The care and use of animals was carried out in accordance with UK Home Office regulations and the Animal Care and Use Committee of the University of Padua. Inner Ear Clearing and Ossicle Dissection Half heads were fixed in Bodian’s fixative and cleared with glycerol using a standard protocol. The inner ear was examined for signs of malformation. Middle ear ossicles were dissected out and studied. Six Obl/+ mutants and six littermate controls between 3–4 months age were analysed. Scanning Electron Microscopy Inner ears were fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, the organ of Corti was exposed and samples were processed using the osmium tetroxide-thiocarbohydrazide (OTOTO) method [37]. After critical point drying and sputter coating with gold, samples were examined using a Phillips XL30 scanning electron microscope at 10 kV or a Hitachi S4800 FE Scanning Electron Microscope at 5 kV. Initial characterisation was performed on 3 Obl/+ and 3 littermate controls at 3–4 months of age. Hair cell degeneration was assessed in the basal turn (20– 30% of the total distance from the base of the cochlear duct) and PLoS Genetics | www.plosgenetics.org 7 October 2008 | Volume 4 | Issue 10 | e1000238