Immunofluorescence analysis of Phospho-Tau pThr212 Antibody was done on 70% confluent log phase SHSY5Y cells. The cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.25% Triton™ X-100 for 10 minutes, and blocked with 5% BSA for 1 hour at room temperature. The cells were labeled with Phospho-Tau pThr212 Antibody at 1ug/mL in 1% BSA and incubated for 3 hours at room temperature and then labeled with Alexa flour 488 Goat Anti-Rabbit IgG Secondary Antibody (A11008) at a dilution of 1:400 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI. F-actin (Panel c: red) was stained with Alexa Fluor 594 Phalloidin. Panel d is a merged image showing nuclear and cytoplasmic localization. Panel e is a no primary antibody control. The images were captured at 40X magnification.
Flow cytometry analysis of Tau [pT212] was done on SH-SY5Y cells. Cells were fixed with 70% ethanol for 10 minutes, permeabilized with 0.25% Triton™ X-100 for 20 minutes, and blocked with 5% BSA for 30 minutes at room temperature. Cells were labeled with Tau [pT212] Rabbit Polyclonal Antibody (red histogram) or with rabbit isotype control (pink histogram) at 3-5 ug/million cells in 2.5% BSA. After incubation at room temperature for 2 hours, the cells were labeled with Alexa Fluor® 488 Goat Anti-Rabbit Secondary Antibody at a dilution of 1:400 for 30 minutes at room temperature. The representative 10,000 cells were acquired and analyzed for each sample using an Attune® Acoustic Focusing Cytometer. The purple histogram represents unstained control cells and the green histogram represents no-primary-antibody control.
Western blot analysis of Tau [pT212] was performed by loading 20 ug of Mouse Brain (lane1) and Rat Brain (lane2) tissue lysate using Novex® NuPAGE® 4-12 % Bis-Tris gel (NP0322BOX), XCell SureLock™ Electrophoresis System (EI0002), Novex® Sharp Pre-Stained Protein Standard (LC5800), and iBlot® Dry Blotting System (IB21001). Proteins were transferred to a nitrocellulose membrane and blocked with 5 % skim milk for 1 hour at room temperature. Tau [pT212] was detected at ~ 42 kDa using Tau [pT212] Rabbit polyclonal Antibody at 1:1000 in 5 % skim milk at 4°C overnight on a rocking platform. Goat Anti-Rabbit IgG - HRP Secondary Antibody at 1:5000 dilution was used and chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (WP20005).
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