Anti tmem240 antibody

Two different fixation procedures were used for the electron microscopy analysis: LR White embedding gives higher sensitivity, whereas Araldite embedding preserves cellular structures. The cerebellum was rapidly dissected, incubated in fixation buffer at 4 °C, and post-fixed in 1% osmic acid, 0.1 M phosphate buffer. The sections were dehydrated using a series of increasing ethanol concentrations, impregnated with 96% LR White resin/ethanol or Araldite/absolute ethanol, and then stored in pure buffer at 4 °C. After polymerization (4 °C under UV for LR White or 56 °C for Araldite), the sections (85-nm ultra-thin sections) were incubated with the anti-TMEM240 antibody (Santa Cruz Biotechnology) for three days in 0.1 M Tris, 0.15 M NaCl, 1% BSA, pH 7.4, and 1% normal donkey serum, supplemented (for Araldite embedding) with 30% H O . The same buffer was used for washing. The sections were then incubated with donkey anti-goat antibodies conjugated to 12-nm gold particles (in 0.1 M Tris, 0.5 M NaCl, 1% BSA, pH 7.4, and normal donkey serum 1%) for 1 h. After washing with the same buffer, the contrast was developed by exposure to 2% uranyl acetate in 50% ethanol for 10 s (LR White embedding) or 2% uranyl acetate and then lead citrate for 8 min each (Araldite embedding). The sections were visualized under a Zeiss EM 900 microscope at magnifications from × 3000 to × 140,000.