Leo em910

For EM experiments, 5 μL of 10 μM tau-K18 fibril reactions were placed on 400 mesh Formvar/Carbon film-coated copper grids (Ted Pella, Inc.) for 5 min, quickly washed with distilled water two times, and then stained with 2% uranyl acetate for 30 sec. A LEO EM910 transmission electron microscope (Carl Zeiss Microscopy) at an acceleration voltage of 80 kV was used to visualize the fibrils. The camera used was an Orius SC1000 CCD Camera, 2672 × 4008 pixels (11 megapixels) and Digital Micrograph version 2.3 software was used to acquire the images (Gatan, Inc.). A minimum of N = 10 independent regions of each grid was imaged from N = 3 independent replicates of WT, K280Q, or P301L mutant fibrils.

Histological examinations of the kidney biopsies at the age of 28 and 32 years were performed with light microscopy of sections stained with Periodic acid-Schiff (PAS) reaction and hematoxylin and eosin staining, immunohistochemistry and electron microscopy. Immunohistochemistry of the latest biopsy was performed with antibodies specific for IgA, IgG, IgM, C1q, and C3c (all polyclonal rabbit antibodies, Dako; Dilution and Code No. IgA 1:200000, A0262, IgG 1:150000, A0423, IgM 1:100000, A0425, C1q 1:100000, A0136, C3c 1:100000, A0062) after digestion with PronaseE for 45 min. For detection Envision Kit (Dako) was applied and DAB was used as a chromogen. For electron microscopy fixed renal biopsies were dehydrated and embedded in Epon. Semithin and ultrathin sections were prepared and stained with methylene blue or uranyl acetate/lead citrate, respectively. Ultrathin sections were then analyzed with a Zeiss electron microscope LEO EM 910 or LEO EM 912 (Zeiss, Oberkochen, Germany) at various magnifications. Histological and electron microscopic images were only available from the latest kidney biopsy.