Envision dual link hrp

The cells were fixed with 4% formaldehyde diluted in PBS from 37% stock solution (Sigma), and permeabilized with 0.5% saponin (Sigma) and 0.3% Triton X-100 (Sigma) in PBS. Following the blocking step for 1 h (blocking solution, 10% FBS and 0.1% Triton X-100 in PBS), the cells were incubated with phospho-Smad3 (Ser423/425) antibody in PBS containing 2% FBS and 0.1% Triton X-100 solution for 1 h. After extensive washing with PBS, the coverslips were incubated for 30 min with EnVision + Dual Link-HRP (Dako), followed by DAB detection solution (Dako) for a few minutes at room temperature. Finally, after being rinsed with purified water and counterstained with hematoxylin (Sigma) for 3-4 min, the coverslips were mounted on glass microscopic slides using 90% glycerol and visualized using a light microscope. The percentage of nuclear p-Smad3 staining was calculated as the number of p-Smad3-positive nuclei over the total number of hematoxylin-stained nuclei counted in four random microscope fields (magnification, 200 ×).

7 m paraffin sections were treated in 1% hydrogen peroxide in methanol followed by 10% normal horse serum and incubated in the monoclonal antibody AT8 AntiHuman Phos PHF Tau pSer202/Thr 205 (Thermo MN1020) at 1:1000 for 60 min at 37 • C. Slides were incubated with Envision Dual Link HRP (Dako K4061) at room temperature for 30 min and visualized with 3,3 diaminobenzidine tetrahydrochloride (MP Biomedicals AF312215). Sections were counterstained with Improved Harris hematoxylin and viewed under bright-field microscopic illumination. The same grid-based system as above was used to count the number of LC neuronal cell bodies that contained tau HYP .