{"id":125,"date":"2015-08-03T13:15:21","date_gmt":"2015-08-03T04:15:21","guid":{"rendered":"http:\/\/www-p.cc.ocha.ac.jp\/bio-shimada-lab\/?page_id=125"},"modified":"2017-12-12T14:58:45","modified_gmt":"2017-12-12T05:58:45","slug":"pr_02microscope","status":"publish","type":"page","link":"https:\/\/www-p.sci.ocha.ac.jp\/bio-shimada-lab\/ex_protocol\/pr_02microscope.html","title":{"rendered":"\u9855\u5fae\u93e1\u95a2\u4fc2"},"content":{"rendered":"

<\/a>\u5149\u5b66\u9855\u5fae\u93e1\u306e\u4f7f\u3044\u65b9<\/h2>\n

\u30d1\u30bd\u30b3\u30f3\u306e\u96fb\u6e90\u3092\u5165\u308c\u308b<\/p>\n

PW\u306a\u3057<\/p>\n

\u5de6\u4e0a\u304b\u30894\u756a\u76ee\u306eAxioVision\u3092\u30c0\u30d6\u30eb\u30af\u30ea\u30c3\u30af<\/p>\n

BF(\u5de6\u5074)\u306e\u4e0a\u304b\u30892\u756a\u76ee\u306e\u30e9\u30a4\u30d6\u3092\u30af\u30ea\u30c3\u30af<\/p>\n

\u9732\u51fa\u3001\u30db\u30ef\u30a4\u30c8\u30d0\u30e9\u30f3\u30b9\u3092\u8abf\u6574<\/p>\n

\u30e9\u30a4\u30d6\u306e\u30d7\u30ed\u30d1\u30c6\u30a3\u3067\u3055\u3089\u306b\u8abf\u6574<\/p>\n

\u76ee\u5b89\u3068\u3057\u3066\u30fb\u30fb\u30fb 40\u500d\u3001\u5149\u91cf9\u3067 \u7dd1\u85fb \u30c7\u30a3\u30b9\u30d7\u30ec\u30fc\u3000-0.53, 1.11, 0.40 \u8abf\u6574\u300013ms, 134% \u8272\u8abf\u30000.77, 1.30, 0.93<\/p>\n

\u30b9\u30b1\u30fc\u30eb\u3092\u30c1\u30a7\u30c3\u30af(\u500d\u7387\u3092\u5909\u3048\u305f\u3068\u304d\u306f\u30b9\u30b1\u30fc\u30eb\u3082\u5909\u3048\u308b)<\/p>\n

\u30b9\u30ca\u30c3\u30d7<\/p>\n

\u30b9\u30b1\u30fc\u30eb\u30d0\u30fc\u3092\u5165\u308c\u308b<\/p>\n

\u4fdd\u5b58<\/p>\n

\u30ab\u30e1\u30e9\u306f\u5b9f\u4f53\u3067\u3082\u5012\u7acb\u3067\u3082\u4f7f\u7528\u3067\u304d\u308b\u304c\u3001\u79fb\u52d5\u3055\u305b\u30de\u30a6\u30f3\u30c8\u304c\u7570\u306a\u308b\u306e\u3067\u6ce8\u610f!!!<\/p>\n

\n

 <\/p>\n

\u86cd\u5149\u89b3\u5bdf<\/h2>\n

DAPI\u3067\u6838\u67d3\u8272<\/h4>\n
    \n
  1. \u9162\u9178:\u30a8\u30bf\u30ce\u30fc\u30eb=1:3\u3067\u6df7\u5408(1.5ml\u30a8\u30c3\u30da\u30f3\u306b\u9162\u9178200\u03bc:\u30a8\u30bf\u30ce\u30fc\u30eb600\u03bc)\u3092\u4f5c\u308a\u3001\u305d\u3053\u306b\u85fb\u4f53\u3092\u5165\u308c\u308b\u30002\u6642\u9593\u5f85\u3064\u300070%\u30a8\u30bf\u30ce\u30fc\u30eb\u306b\u7f6e\u63db\u3057\u51b7\u8535\u5eab\u3067\u4fdd\u7ba1<\/li>\n
  2. 70%\u30a8\u30bf\u30ce\u30fc\u30eb\u3092\u6368\u3066\u3066\u3001DW\u30673\u56de\u6d17\u3046<\/li>\n
  3. \u30ed\u30b7\u30e5\u88fd\u54c1\u306eDAPI(10mg\u3092DW5ml\u3067\u6eb6\u304b\u3057\u3066-20\u2103\u4fdd\u5b58)\u3092DW\u30672000\u500d\u306b\u8584\u3081\u3066\u4f7f\u7528(2mlDW\u306b1\u00b5lDAPI\u6db2)\u30025\u5206\u5f85\u3064\u3002<\/li>\n
  4. UV\u3067\u89b3\u5bdf<\/li>\n<\/ol>\n

    \u30df\u30b9\u30e9\u30de\u30a4\u30b7\u30f3\u3067\u6838\u67d3\u8272<\/h4>\n
      \n
    1. \u9162\u9178:\u30a8\u30bf\u30ce\u30fc\u30eb=1:3\u3067\u6df7\u5408(1.5ml\u30a8\u30c3\u30da\u30f3\u306b\u9162\u9178200\u03bc:\u30a8\u30bf\u30ce\u30fc\u30eb600\u03bc)\u3092\u4f5c\u308a\u3001\u305d\u3053\u306b\u85fb\u4f53\u3092\u5165\u308c\u308b\u30002\u6642\u9593\u5f85\u3064\u300070%\u30a8\u30bf\u30ce\u30fc\u30eb\u306b\u7f6e\u63db\u3057\u51b7\u8535\u5eab\u3067\u4fdd\u7ba1<\/li>\n
    2. Buffer\u306e\u4f5c\u88fd(0.1M \u30af\u30a8\u30f3\u91781.78ml\u30680.2M Na2PO4 8.22ml\u3092\u30b3\u30cb\u30ab\u30eb\u30c1\u30e5\u30fc\u30d6\u3067\u6df7\u5408\u3057\u3001MgCl2\u30fbH2O 24mg\u3092\u5165\u308c\u308b)<\/li>\n
    3. 70%\u30a8\u30bf\u30ce\u30fc\u30eb\u3092\u6368\u3066\u3066\u3001DW\u30673\u56de\u6d17\u3046<\/li>\n
    4. Buffer\u3092400\u03bc\u5165\u308c\u3001\u305d\u3053\u306b\u30df\u30b9\u30e9\u30de\u30a4\u30b7\u30f3(250\u03bcg\/ml\u306b\u8abf\u6574\u6e08\u307f)\u3092100\u03bc\u5165\u308c\u308b<\/li>\n<\/ol>\n

      \u751f\u4f53\u3067\u306e\u7d30\u80de\u58c1\u67d3\u8272<\/h4>\n
        \n
      1. 0.01% Fluorescent Brightener 28 \u6d77\u6c34\u6eb6\u6db2\u3092\u4f5c\u308a\u3001\u30aa\u30fc\u30c8\u30af\u30ec\u30fc\u30d6\u3000\u3000(FB 20mg+SW 200ml)<\/li>\n
      2. \u85fb\u4f53\u3092\u7dba\u9e97\u306b\u6d17\u6d44\u3057\u3001\u30b7\u30e3\u30fc\u30ec\u306b\u3044\u308c\u308b\u3002<\/li>\n
      3. \u9069\u5f53\u91cfFB\u6d77\u6c34\u6eb6\u6db2\u3092\u5165\u308c\u300130-60mjn\u5f85\u3064\u3002<\/li>\n
      4. 2-3\u56de\u3001\u6ec5\u83cc\u6d77\u6c34\u3067\u6d17\u6d44<\/li>\n
      5. UV\u4e0b\u30671\u3064\u3065\u3064\u89b3\u5bdf<\/li>\n
      6. 24\u7a74\u30b7\u30e3\u30fc\u30ec\u306b\u5165\u308c\u57f9\u990a<\/li>\n
      7. 1\u65e5\u304a\u304d\u306b\u89b3\u5bdf<\/li>\n<\/ol>\n

        FDA\u67d3\u8272\u751f\u5b58\u7387\u30ab\u30a6\u30f3\u30c8\u6cd5<\/h4>\n

        \u7d30\u80de\u5185\u306b\u53d6\u308a\u8fbc\u307e\u308c\u305f\u7121\u8272\u900f\u660e\u306eFDA\u306f\u3001\u30a8\u30b9\u30c6\u30e9\u30fc\u30bc\u306b\u3088\u308a\u5206\u89e3\u3055\u308c\u3001\u7d2b\u5916\u7dda\u306b\u3088\u308a\u86cd\u5149\u3092\u767a\u3059\u308b\u3002\u3053\u308c\u3092\u86cd\u5149\u9855\u5fae\u93e1\u30d6\u30eb\u30fc\u52b1\u8d77\u3067\u89b3\u5bdf\u3057\u3066\u3001\u7d30\u80de\u306e\u751f\u6b7b\u3092\u5224\u5b9a\u3059\u308b\u65b9\u6cd5\u3002\u9ad8\u7b49\u690d\u7269\u306a\u3069\u3067\u3088\u304f\u7528\u3044\u3089\u308c\u3066\u3044\u308b\u3002<\/p>\n

        <\u624b\u9806><\/h4>\n
          \n
        1. FDA 5mg\u3092100%\u30a2\u30bb\u30c8\u30f31ml\u306b\u6eb6\u304b\u3057\u3001\u30a8\u30c3\u30da\u30f3\u30c1\u30e5\u30fc\u30d6\u306b (50\u03bcl\u305a\u3064) \u5206\u6ce8\u3002\u3053\u306e\u30b9\u30c8\u30c3\u30af\u67d3\u8272\u6db2\u306f\u3001\u30d5\u30ea\u30fc\u30b6\u30fc (-20\u2103) \u5185\u3067\u4fdd\u7ba1\u3057\u3066\u304a\u304f\u3002<\/li>\n
        2. \u4f7f\u7528\u76f4\u524d\u3001\u30b9\u30c8\u30c3\u30af\u67d3\u8272\u6db25\u03bcl\u3001\u57f9\u5730495\u03bcl\u3092\u30a8\u30c3\u30da\u30f3\u30c1\u30e5\u30fc\u30d6\u4e2d\u3067\u6df7\u5408\u3057\u3001100\u500d\u306b\u5e0c\u91c8\u3059\u308b\u3002<\/li>\n
        3. \u8a66\u6599\u3092\u67d3\u8272\u6db2\u306b\u5165\u308c\u3001\u5ba4\u6e29\u306720\u5206\u7f6e\u3044\u305f\u5f8c\u3001\u86cd\u5149\u9855\u5fae\u93e1\u3067\u89b3\u5bdf\u3059\u308b\u3002<\/li>\n
        4. \u30ce\u30de\u30eb\u30b9\u30ad\u30fc\u306eU?DICT\u3092\u306f\u305a\u3059\u3002\u86cd\u5149\u30df\u30e9\u30fc\u306fB\u52b1\u8d77\u306e\u72ed\u57df\u300cNIBA\u300d\u3067\u3082\u5e83\u57df\u300cWIB\u300d\u3067\u3082OK\u3002\u5e83\u57df\u300cWIB\u300d\u306e\u65b9\u304c\u8449\u7dd1\u4f53\u306e\u81ea\u5bb6\u86cd\u5149\u304c\u898b\u3048\u3066,\u6b7b\u7d30\u80de\u304c\u30ab\u30a6\u30f3\u30c8\u3057\u3084\u3059\u3044\u3002<\/li>\n<\/ol>\n
          \n

           <\/p>\n

          TEM\u3067\u306e\u89b3\u5bdf<\/h2>\n
            \n
          • \u524d\u56fa\u5b9a\u3000\u30004\u2103\u30002\u6642\u9593<\/li>\n<\/ul>\n
              \n
            1. 10%\u30b0\u30eb\u30bf\u30fc\u30eb\u30a2\u30eb\u30c7\u30d2\u30c9\u3000100ml25%\u30b0\u30eb\u30bf\u30fc\u30eb\u30a2\u30eb\u30c7\u30d2\u30c9 40ml\u306bDW\u300060ml\u52a0\u3048\u305f<\/li>\n
            2. 0.2M\u30ab\u30b3\u30b8\u30eb\u9178Na(\u6bd2\u7269\u4fdd\u7ba1\u5eab)+0.2% CaCl2(+\u6d77\u7523\u7a2e\u306e\u5834\u5408,4%NaCl) 0.2M\u3000100ml<\/li>\n<\/ol>\n

              \u30d3\u30fc\u30ab\u30fc\u306b50ml\u3000DW, \u30ab\u30b3\u30b8\u30eb\u9178Na\u30004.28g\u5165\u308c,\u6eb6\u3051\u305f\u3089,0.2% CaCl2\u306b\u306a\u308b\u3088\u3046\u306b200mg\u52a0\u3048,pH7.2\u306b\u5408\u308f\u305b,100ml\u306bfill up(+\u6d77\u7523\u7a2e\u306e\u5834\u5408, NaCl 4g\u3092\u6700\u5f8c\u306b\u52a0\u3048\u308b)<\/p>\n

              \u30c7\u30a3\u30b9\u30dd\u30b7\u30e3\u30fc\u30ec\u3082\u3057\u304f\u306f\u30a8\u30c3\u30da\u30f3\u30c1\u30e5\u30fc\u30d6\u306b\u2460\u30925ml\u5165\u308c,\u305d\u3053\u306b\u2461\u309210ml\u5165\u308c,\u305d\u3053\u306b\u57f9\u990a\u3057\u3066\u3044\u308b\u6c34(\u6d77\u6c34,\u6c7d\u6c34,\u6de1\u6c34)\u3092\u540c\u91cf\u306e5ml\u5165\u308c,\u305d\u3053\u306b\u85fb\u4f53\u3092\u5165\u308c\u308b\u3002\u305d\u306e\u5f8c,\u85fb\u4f53\u30925mm\u89d2\u306b\u5c0f\u3055\u304f\u3059\u308b\u3002<\/p>\n

              \u6d77\u7523\u306e\u5834\u5408,30\u5206\u5f8c,\u30a8\u30c3\u30da\u30f3\u30c1\u30e5\u30fc\u30d6\u306b\u2460\u3092250\u03bc,\u2461\u3092500\u03bc,DW\u3092250\u03bc\u5165\u308c,\u6df7\u305c,\u305d\u3053\u306b,\u5207\u3063\u305f\u85fb\u4f53\u3092\u5165\u308c\u308b\u3002<\/p>\n

              (\u6700\u7d42\u6fc3\u5ea6:2.5%\u30b0\u30eb\u30bf\u30fc\u30eb\u30a2\u30eb\u30c7\u30d2\u30c9,0.1M\u30ab\u30b3\u30b8\u30eb\u9178Na,0.1% CaCl2(+\u6d77\u7523\u7a2e\u306e\u5834\u5408,2%NaCl))<\/p>\n

                \n
              • \u6d17\u6d44 10\u5206\u6bce\u30006-8\u56de 0.1% CaCl2\u3000 0.1M \u30ab\u30b3\u30b8\u30eb\u9178\u7de9\u885d\u6db2\u3000(+\u6d77\u7523\u7a2e\u306e\u5834\u5408,2%NaCl) \u5ec3\u6db2\u306f\u30ab\u30b3\u30b8\u30eb\u9178\u5ec3\u6db2\u30d3\u30f3\u3078<\/li>\n
              • \u30a2\u30eb\u30c7\u30d2\u30c9\u306fOsO4\u306e\u9084\u5143\u5264\u3068\u306a\u308a\u3046\u308b\u306e\u3067\u6d17\u6d44\u306f\u5341\u5206\u306b\u884c\u306a\u3046\u3002<\/li>\n
              • \u5f8c\u56fa\u5b9a 4\u2103\u30002\u6642\u9593 \u6d17\u6d44\u6db2\u3092\u629c\u304d,\u7d0420\u03bc\u6b8b\u3057(\u5168\u91cf\u53d6\u3063\u305f\u5834\u5408\u306f\u52a0\u3048),\u305d\u3053\u306b4%OsO4\u3092\u5c02\u7528\u30d4\u30da\u30c3\u30c8\u3067\u5165\u308c\u308b\u3002<\/li>\n<\/ul>\n

                (\u6700\u7d42\u6fc3\u5ea6:\u30001-2% OsO4,0.1% CaCl2,0.1M \u30ab\u30b3\u30b8\u30eb\u9178\u7de9\u885d\u6db2)<\/p>\n

                OsO4\u306f\u63ee\u767a\u6027\u306e\u9ad8\u3044\u6bd2\u7269!\u5fc5\u305a,\u624b\u888b\u3092\u3057\u3066\u30c9\u30e9\u30d5\u30c8\u5185\u3067\u4f5c\u696d\u3059\u308b\u3053\u3068\u3002<\/p>\n

                \u5ec3\u6db2\u56de\u53ce\u304c\u3067\u304d\u306a\u3044\u305f\u3081,\u5fc5\u8981\u91cf\u306e\u307f\u4f7f\u3046\u3053\u3068\u3002<\/p>\n