• 我要登录|
  • 免费注册
    |
  • 我的丁香通
    • 企业机构:
    • 成为企业机构
    • 个人用户:
    • 个人中心
  • 移动端
    移动端
丁香通 logo丁香实验_LOGO
搜实验

    大家都在搜

      大家都在搜

        0 人通过求购买到了急需的产品
        免费发布求购
        发布求购
        点赞
        收藏
        wx-share
        分享

        immunocytochemistry

        互联网

        1058

         

        A. Plating:

        To sterilize glass coverslips, dip in ethanol and flame.
             We use 22x22x1 mm3 coverslips and put them in 6-well plates.
        Seed 100,000 cells per well overnight and fix the next day.

        B. Fixation:

        Remove the media and rinse once with PBS.
        Remove the PBS and immediately add -20°C methanol. (Do not allow the cells to dry.)
        Put the plate in a -20°C freezer for 5 min.
        Remove the methanol and add PHEM buffer. Fixed cells are kept at 4°C in PHEM.

        C. Antibody incubation:

        Block with appropriate sera (2.5 to 5%) in PHEM buffer for 1 hr with gentle rocking.
        Add primary antibody to the blocking buffer and incubate for 1 hr with gentle rocking.
        Remove and wash 4 x 10 min with PHEM buffer.
        Add secondary antibody in PHEM buffer with sera and incubate for 30 min with gentle rocking.
        Remove and wash 4 x 10 min with PHEM buffer.

        D. Mounting:

        Pick up coverslip with forceps and drain away excess buffer (can gently aspirate if desired).
        Put ~20 μl "antifade" on slide and gently lay coverslip on top.
        After removing excess antifade, either by blotting with Kimwipe or aspirating, seal with Sally
        Hansen clear nail polish. (This brand supposedly works better than others.)
        KEEP IN THE DARK AT ALL TIMES.
        Store in -20°C freezer.

        Solutions:

        PHEM buffer:
        25 mM HEPES
        10 mM EGTA
        60 mM PIPES
        2 mM MgCl2
        pH = 6.9
        (Add in this order.)
        Antifade: 1 ml
        1 mg p-phenylene diamine hydrochloride
        Dissolve in 0.1 ml 10x PBS (20 min at RT)
        Add 0.9 ml 100% glycerol
        Keep covered at all times and no vortexing.
        If it turns brown, it’s no good.
        Aliquot and store at -70°C.

         

        ad image
        提问
        扫一扫
        丁香实验小程序二维码
        实验小助手
        丁香实验公众号二维码
        扫码领资料
        反馈
        TOP
        打开小程序