Ehrlichia secretes Etf-1 to induce autophagy and capture nutrients for its growth through RAB5 and class III phosphatidylinositol 3-kinase

Lin, Mingqun; Liu, Hongyan; Xiong, Qingming; Niu, Hua; Cheng, Zhihui; Yamamoto, Akitsugu; Rikihisa, Yasuko

The immunogold labeling method with preembedding gold enhancement was performed as described. 132 In brief, cells were fixed in 4% PFA in 0.1 M sodium phosphate buffer (pH 7.4) for 2 h at room temperature and permeabilized in the same buffer containing 0.25% saponin for 30 min. After blocking for 30 min in the same buffer containing 0.1% saponin, 10% bovine serum albumin, 10% normal goat serum (Vector Laboratories, S-1000), and 0.1% cold-water fish skin gelatin (Sigma, G7765) (blocking solution), specimens were incubated with mouse anti-GFP or control mouse IgG overnight, followed by incubation with colloidal gold (1.4 nm in diameter; Nanoprobes, 2001) conjugated to goat anti-mouse IgG in blocking solution for 2 h. After treatment with a silver enhancement kit (SilverEnhance EM; Nanoprobes, 2012) for 2 min at room temperature to intensify the signal, the specimens were postfixed in 1% OsO 4 solution containing 1.5% potassium ferrocyanide, dehydrated in a series of graded ethanol solutions, and embedded in epoxy resin. Ultra-thin sections were collected, stained with uranyl acetate and lead citrate, and observed with a Hitachi H7600 (Tokyo, Japan) transmission electron microscope.