(d) An example of p-tau (Ser369/Ser404 phosphorylation-dependent epitopes accessed by PHF-1 antibody) signal in a synaptosomal fraction from normal human brain cortex: 9.4 % of p-tau positive events with average intensity 17.2 RFU. (Subheading 3.1, step 2 2 and Cardiogenol C hydrochloride Note 7). Homogenize tissue using a Teflon/glass homogenizer (clearance 0.1C0.15 mm) by eight gentle up and down strokes at 800 rpm. Save 200C500 l of homogenate for P2-quality assay ((4 C) to pellet P2 fraction. Decant the supernatant and resuspend the pellet in fixation solution, incubate for 1 h at 4 C. Centrifuge the sample for 4 min at 4000 (4 C) to pellet P2 fraction. Decant supernatant. Resuspend fixed P2 fraction in 500 l permeabilization buffer ((4 C) to pellet P2-fraction. Decant supernatant. Steps 10(steps 7 8 (4 C). Repeat the washing step 2 2 times, protect from light. Resuspend immunostained P2-fraction in Mouse monoclonal to BRAF washing buffer and transfer to FACS tubes, protected from light. 3.4 Flow Cytometry (FC) Analysis ( em laser alignment /em , em laser time delay etc. /em ) em is beyond the scope of this chapter /em . Run size calibration standards, we use polystyrene beads (0.75, 1.5, and 4.5 m) and adjust the forward scatter (FSC) and the side scatter (SSC) detectors to place the populations of interest (synaptosome sizes are between 0.75 and 1.5 m) on scale, adjust FSC threshold to exclude noise and apply FSC-based size gate (Fig. 2a). Open in a separate window Fig. 2 Flow cytometry analysis of synaptosomal preparations. (a) An example of size standard acquisition for FSC-based size gate establishment. (b) Background labeling in presence of isotype control antibodies ( em negative control /em ): only 1 1.8 % from the total 10,000 events collected within applied size gates show positive signal with average intensity 46.3 relative fluorescence units (RFU). (c) Presynaptic marker (SNAP25)-positive events Cardiogenol C hydrochloride within the size gate ( em positive control /em ): 97.6 % positive events with average intensity 345.8 RFU. (d) An example of p-tau (Ser369/Ser404 Cardiogenol C hydrochloride phosphorylation-dependent epitopes accessed by PHF-1 antibody) signal in a synaptosomal fraction from normal human Cardiogenol C hydrochloride brain cortex: 9.4 % of p-tau positive events with average intensity 17.2 RFU. (e) An example of p-tau (PHF-1) signal in a synaptosomal preparation from brain of a subject with a late stage of Alzheimers disease: 42.5 % of p-tau positive events with average intensity 76.9 RFU Run controls: unstained samples (blanks), negative (isotype) controls, and positive controls (samples labeled with antibodies against presynaptic markers; we routinely use SNAP25, synaptophysin or VGluT1 antibodies) and samples of interest to confirm and/or optimize the settings by adjusting voltage on corresponding detectors (Fig. 2b, c and Note 15). Adjust fluorescent compensation by running single and multi-labeled samples, if multiple color data will be collected from each sample. Collect from 5000 to 10,000 events within the size gate from each experimental sample. Analyze the data ( em see /em Fig. 2d, e for an example). Footnotes 1EDTA and EGTA compounds, especially in the free acid form, are almost insoluble in water. Place magnet stirring bar to the EDTA (EGTA) water mixture and place the beaker on the magnet stirrer hot plate. Stir constantly, carefully titrate with sodium hydroxide (10 N NaOH) and continuously measure pH. Stop when pH reaches 8.0 and continue stirring until EDTA/EGTA is fully dissolved in water. 2Sucrose solution without protease/phosphates inhibitors can be made in advance and either kept on ice or frozen at ?20 C for prolonged storage. Immediate cryopreservation of human brain autopsy samples is very important in order to minimize tissue degradation and obtain high quality synaptosomal fraction, thus all of the feasible progress planning ought to be made to have the ability to begin cryopreservation procedure when the autopsy examples arrive towards the laboratory. 3To standardize PBS composition we use commercially obtainable PBS.