This study also highlights the fundamental difference between tau physiological cell-to-cell transfer and tau pathological propagation. with cold 0.9% NaCl followed by cold 4% PFA for 20?min. The brains were immediately removed, fixed overnight in 4% PFA, placed in 20% sucrose for 24?h and frozen until further use. Free-floating coronal cryostat sections (40?m thickness) were used for immunohistochemical analysis. For RNA extraction, 2?weeks post-injection, were deeply anesthetised (pentobarbital 50?mg/kg). Brains were dissected, and 1-mm-thick coronal sections were generated using an acrylic rat brain matrix (Electron Microscopy Sciences). The sections were immediately frozen on dry ice and stored at ??80?C until further use. Immunohistochemistry For rats, immunohistochemistry procedures were previously described . Briefly, sections from the entire brain were washed in PBS-0.2% Triton X-100 and treated for 30?min with H2O2 (0.3%). Non-specific binding was blocked using goat serum (1:100 in PBS, Vector) for 60?min. Sections were incubated overnight at 4?C with the monoclonal antibodies AT8 (Thermo Scientific; MN1020C1:400; phosphorylated residues 202, 205 and 208 of tau) , ADx-215 [10, 54] (1:10,000; human specific total tau) or MC1/Alz50 (kind gifts from Dr. Peter Davies C 1:10,000; misfolded tau) in PBS-0.2% Triton X-100. After several washes, labelling was amplified by incubation with an anti-mouse biotinylated IgG (1:400 in PBS-0.2% Triton X-100, Vector) for 60?min followed by the application of the ABC kit (1:400 in PBS, Vector) prior to visualization with 0.5?mg/ml DAB (Vector) in Tris-HCl 50?mmol/L, pH?7.6, containing 0.075% H2O2. Brain sections were counter-stained in a cresyl violet solution (0.5%) and mounted with Vectamount (Vector) for microscopic analysis. For human sections, 9?m thick paraffin-embedded sections of hippocampus, Iopromide temporal cortex and visual cortex of 10 human cases (Table?1) were cut using a microtome and placed on glass slides. Slides were incubated at 55?C for 4?h before being immerged in successive 8?min baths of xylene twice, EtOH 100% twice, EtOH 95%, EtOH 70%, EtOH 50% and PBS three times. Slides were then incubated in boiling citrate buffer (citric acid Iopromide anhydrous 10?mM, Tween20 0.05%, pH?=?6) in a microwave at low power for 20?min. Slides were immerged in Tris-Buffered Saline (TBS) with 0.5% triton X-100 for 30?min followed by blocking with TBS, 10% Normal Goat Iopromide Serum for 1?h. Slides were incubated overnight at 4?C with primary antibodies (Alz50, kind gift of Dr. Peter Davis: 1/50 and AT8 1/400) in TBS, 5% NGS, 0.05% Triton X-100. Slides were washed 4 times with TBS and then incubated with secondary antibodies (anti-mouse IgM 568 and anti-mouse IgG 488 1/400, Invitrogen) diluted in TBS, 5% NGS. Slides were washed 4 times with TBS and counterstained with Sudan black (0.1% in 70% EtOH, filtered) for 20?min. Slides were washed 4 times with TBS and coversliped with Fluoromount G with Dapi (Thermo Fisher Scientific). Slides were scanned using an Olympus VS-120 slide scanner and then 100% of neurons were counted using the cellSens software. All human tissues come from the Lille Neurobank and the Massachusetts Alzheimers Disease Research center and written consent forms have been obtained accordingly to the local legislations and ethical committees. Human brains extracts were obtained from the Massachusetts Alzheimers Disease Research Center (grant number P50 AG005134, under IRB protocol 1999P003693) and the Lille Neurobank (CRB/CIC1403 Biobank, BB-0033-00030, agreement DC-2008-642) fulfilling criteria of the local laws and regulations on biological resources with donor consent, data protection and ethical committee review. Table 1 Human case demographics Male, Female, Post Mortem interval, genetic FrontoTemporal Lobar Dementia-Tau, Alzheimers Disease, values have been determined using one-way ANOVA tests and a Tukey post-hoc test or a Pearsons Chi-squared test with Yates continuity correction as indicated in the figure legend. Differences were considered to be statistically significant if p?0.05. Results Differential misfolding/hyperphosphorylation profile in human MAPT mutant carriers compared to sporadic AD We hypothesized that the mechanisms of tau deposition Iopromide are different in sporadic tauopathies than when a mutation of MAPT gene is present. Therefore, we investigated the presence of tau misfolding and hyperphosphorylation epitopes in human brain samples. We selected six Alzheimers disease patients (two Braak I, two Braak IV, two Braak VI) and 4 patients with Fronto-temporal dementia associated with a mutation of MAPT gene (two P301L, one P332S and one G389R). We stained brain sections from three different regions following the Braak stages: hippocampus, temporal cortex and visual cortex with AT8 antibody for Rabbit Polyclonal to ELOA1 tau hyperphosphorylation and Alz50 for tau misfolding. Three different phenotypes can be observed: neurons positive for both Alz50 and AT8 (Fig.?1a-c, arrows), neurons positive only for AT8 (Fig. ?(Fig.1a-c,1a-c, arrowheads) and, more rarely, neurons positive only for Alz50 (Fig. ?(Fig.1a,1a, star). We quantified in each case the number of neurons in each category. We counted a total number of 27,214 neurons for the AD cases and 15,460 for the mutant cases. Most of the neurons.