015% H2O2 as cosubstrate. Adjacent serial sections were used to directly compare pathological structures INK 128 research buy recognized by antibodies listed in Table 1. For double-label immunofluorescence, sections were blocked with 10% NGS (Sigma) in TBS for 30 min. Double-labelling experiments were conducted by combining two of the primary antibodies listed in Table 1. Bound monoclonal antibodies were detected with FIT-C or TRIT-C conjugated goat anti-mouse IgG (γ-specific) and anti-mouse IgM (μ-specific) (Jackson Immuno-Research laboratories, Bar Harbor, ME, USA). In all experiments, incubation with primary antibodies was done overnight at 4°C, followed by 2 h

at room temperature with the appropriate secondary antibodies. The sections were mounted Fulvestrant concentration in antiquenching medium (Vectashield, Vector Laboratories, Inc., Burlingame, CA, USA). Labelled brain sections were viewed with a 40× Plan-Apochromat on a TCP-SP2 Leica (Heidelberg, Germany) laser scanning-confocal microscope. Additional high power lenses (60× and 100×) were used to critically evaluate colocalization in single optical sections. Confocal images were obtained as single sections and the stack of images was projected as individual two-dimensional extended focus images. Resulting images were analysed using the software included

with the microscope and Image J (Image Processing and Analysis in Java) software. Using the peroxidase technique, NFTs were counted in the area of interest (see Table 2). Morphometric quantification in the areas was assessed on three microscopic fields from randomly chosen regions in the area of interest. Observations were conducted by bright-field microscopy (Nikon FN1, Melville, NY, USA). Identification and counting of pathological structures Phospholipase D1 was conducted using 10× and 20× objective lenses and values expressed per mm2 as previously described [33]. Relative expression intensity was measured in neurones by using Image

J software (Image Processing and Analysis in Java). Values represent relative surface area expression. Student’s t-test was applied when counts were compared between different groups. Statistical analysis was conducted in Excel. Bar diagrams represent the experimental mean; the error bars represent the standard error. For statistical analysis we used the Student’s t-test with the significance set a P-value of 0.05. As mesocortices and the hippocampal formation are the most vulnerable brain areas to NFTs, they were the focus of this study. Mesocortices include entorhinal cortex, perirhinal cortex while the hippocampal formation contains parasubiculum, presubiculum, subiculum, CA1, CA2, CA3, CA4, and dentate gyrus. The same groups of neurones were compared with regard to morphological and cytopathological observations of NFTs for the different tau antibodies. For example, entorhinal layer II was compared in each case with all the tau antibodies. Furthermore, NFTs were compared across areas within each case.