Generated Tg mice expressing V337M human tau. cDNA construct of the V337M human longest tau with myc and FLAG epitope tags at the N and C-terminal ends was generated by PCR-based site-directed mutagenesis.
To estimate tau expression and solubility, brains of Tg mice and non-Tg littermates were carefully extracted. Quantitation and visual analysis of immunoreactivity were performed with a computer-linked LAS-1000 Bio-Imaging Analyzer System (Fuji-film, Tokyo, Japan) using the software program Image Gauge 3.0 (Fujifilm).
The following antibodies were used: mouse monoclonal anti-myc (clone 9E10; Babco), rabbit polyclonal anti-myc (MBL), phosphorylation-independent rabbit polyclonal anti-tau JM, (1:10,000); rabbit polyclonal anti-ubiquitin (Dako, Carpinteria, CA),
and phosphorylation-dependent mouse monoclonal anti-tau AT8, which recognizes phosphorylated tau at Ser202 and Ser205.
Also, phosphorylation-dependent rabbit polyclonal anti-tau PS199 and PS396 (1:400) antibodies which recognize phosphorylation of tau at Ser199 or Ser396 (gift of K. Ishiguro), mouse monoclonal anti-tau antibody Alz50 which recognizes the conformational epitope found in paired helical filaments (gift P. Davies).
Sections intended for confocal laser microscopy were first incubated with either anti-myc, JM, PS199, AT8, or Alz50 antibodies and then incubated with either Alexa488/568-conjugated anti-mouse IgG or Alexa488/568-conjugated anti-rabbit IgG. Subsequent nuclear counterstaining with propidium iodide (PI), a nucleic acid-specific stain, was performed after treatment with RNase. Sections were then examined with a Radiance 2000 KR3 confocal microscope (Bio-Rad).