{"id":13539,"date":"2025-12-04T14:44:51","date_gmt":"2025-12-04T06:44:51","guid":{"rendered":"https:\/\/fanyiming.life\/?p=13539"},"modified":"2025-12-21T01:02:21","modified_gmt":"2025-12-20T17:02:21","slug":"if-protocol","status":"publish","type":"post","link":"https:\/\/fanyiming.life\/?p=13539","title":{"rendered":"IF protocol"},"content":{"rendered":"\n<h2 class=\"wp-block-heading\">Introduction<\/h2>\n\n\n\n<p>IF protocol (before DNA FISH)<\/p>\n\n\n\n<h2 class=\"wp-block-heading\">Materials<\/h2>\n\n\n\n<ul class=\"wp-block-list\">\n<li><strong>0.15% TritonX-100<\/strong><\/li>\n\n\n\n<li>10% TritonX-100 as stock in PBS at RT<\/li>\n\n\n\n<li><strong>0.1% PBS\u2013Tween 20<\/strong><\/li>\n\n\n\n<li>10% Tween 20 as stock in PBS at RT<\/li>\n\n\n\n<li><strong>5% BSA in PBS\u2013Tween20<\/strong><\/li>\n\n\n\n<li>store at 4\u00b0C<\/li>\n<\/ul>\n\n\n\n<h2 class=\"wp-block-heading\">Procedure<\/h2>\n\n\n\n<h3 class=\"wp-block-heading\">Day 1<\/h3>\n\n\n\n<h4 class=\"wp-block-heading\">Prepare working buffer<\/h4>\n\n\n\n<figure class=\"wp-block-table\"><table class=\"has-fixed-layout\"><thead><tr><th>Solution<\/th><th>Component<\/th><th>Volume \/ amount<\/th><th>Final<\/th><\/tr><\/thead><tbody><tr><td><strong>0.15% TritonX-100<\/strong><\/td><td>10% Triton X-100<\/td><td>0.75 ml<\/td><td>0.15% TritonX-100<\/td><\/tr><tr><td><\/td><td>PBS<\/td><td>49.25 ml<\/td><td><\/td><\/tr><tr><td><\/td><td><strong>Total<\/strong><\/td><td><strong>50 ml<\/strong><\/td><td><\/td><\/tr><tr><td><strong>0.1% PBS\u2013Tween 20<\/strong><\/td><td>10% PBS\u2013Tween 20<\/td><td>0.5 ml<\/td><td>0.1% PBS\u2013Tween 20<\/td><\/tr><tr><td><\/td><td>PBS<\/td><td>49.5 ml<\/td><td><\/td><\/tr><tr><td><\/td><td><strong>Total<\/strong><\/td><td><strong>50 ml<\/strong><\/td><td><\/td><\/tr><tr><td><strong>5% BSA in 0.1% PBS\u2013Tween20<\/strong><\/td><td>BSA (W\/V)<\/td><td>0.5 g<\/td><td>5% BSA<\/td><\/tr><tr><td><\/td><td>0.1% PBS\u2013Tween 20<\/td><td>10 ml<\/td><td><\/td><\/tr><\/tbody><\/table><\/figure>\n\n\n\n<h4 class=\"wp-block-heading\">Steps<\/h4>\n\n\n\n<ol class=\"wp-block-list\">\n<li>NCCIT cells should be cultured on cover slips for 2 days (0.3 M, 1.5 Day)<\/li>\n\n\n\n<li>PBS twice<\/li>\n\n\n\n<li>4% PFA (diluted with PBS) 20 min at RT<\/li>\n\n\n\n<li>PBS twice<\/li>\n\n\n\n<li>0.15% TritonX-100 (diluted with PBS) 15 min at RT<\/li>\n\n\n\n<li>PBS twice<\/li>\n\n\n\n<li>5% BSA (diluted with 0.1% PBS\u2013Tween20) 1 h at RT<\/li>\n\n\n\n<li>PBST twice (0.1% PBS\u2013Tween20)<\/li>\n\n\n\n<li>Primary antibody solution (diluted with 0.1% PBS\u2013Tween20) 4 h at RT \/ overnight at 4\u00b0C<\/li>\n<\/ol>\n\n\n\n<h3 class=\"wp-block-heading\">Day 2<\/h3>\n\n\n\n<ol start=\"10\" class=\"wp-block-list\">\n<li>PBST four times<\/li>\n\n\n\n<li>Secondary antibody solution (diluted with 0.1% PBS\u2013Tween20) 2\u20134 h at RT<\/li>\n\n\n\n<li>PBST five times<\/li>\n\n\n\n<li>DAPI 5 min<\/li>\n\n\n\n<li>PBS twice<\/li>\n\n\n\n<li>Seal the coverslips<\/li>\n<\/ol>\n\n\n\n<h4 class=\"wp-block-heading\">Notes<\/h4>\n\n\n\n<ul class=\"wp-block-list\">\n<li>\u4e00\u6297\/\u4e8c\u6297\/DAPI\u2014\u2014180ul\uff0c\u4e8c\u6297\uff081:500\uff09<\/li>\n\n\n\n<li>Seal\u5c01\u7247\u5242\uff0c\u52a0\u7684\u65f6\u5019\u4e0d\u8981\u7b49\u7247\u5b50\u98ce\u5e72\uff0c\u6e7f\u6f09\u6f09\u7684\u65f6\u5019\u5373\u53ef\u5c01\u7247<\/li>\n\n\n\n<li>PBST 2min\/times<\/li>\n<\/ul>\n\n\n\n<p>\u540e\u671f\u62cd\u6444\u7684nd2\u683c\u5f0f\u56fe\u7247\uff08\u5c3c\u5eb7\u663e\u5fae\u955c\u4ea7\u51fa\u7684\u56fe\u7247\u683c\u5f0f\uff09\uff0c\u53ef\u4ee5\u7528FIJI\uff08\u4e00\u79cdimageJ\u7684\u5206\u652f\uff09\u6765\u5904\u7406\uff0c\u8fd9\u91cc\u6709\u4e00\u4e2a<a href=\"https:\/\/fanyiming.life\/?p=11369\" data-type=\"post\" data-id=\"11369\">\u5b8f\u811a\u672c<\/a>\u5f88\u597d\u7528\u3002\u4e0d\u8fc7\u8981\u6ce8\u610fAutoScale\u7b49\u53c2\u6570\uff0c\u5fc5\u987b\u7406\u89e3\u542b\u4e49\u3002<\/p>\n","protected":false},"excerpt":{"rendered":"<p>\u8fd9\u7bc7\u7b14\u8bb0\u8be6\u7ec6\u8bb0\u5f55\u4e86IF protocol\uff08\u514d\u75ab\u8367\u5149\u5b9e\u9a8c\uff09\u7684\u64cd\u4f5c\u6d41\u7a0b\uff0c\u6db5\u76d6\u6750\u6599\u51c6\u5907\u3001\u5de5\u4f5c\u6db2\u914d\u5236\u53ca\u4e3a\u671f\u4e24\u5929\u7684\u5177\u4f53\u6b65\u9aa4\uff0c\u9002\u7528\u4e8eDNA FISH\u524d\u7684\u6837\u672c\u5904\u7406\u3002\u4f5c\u8005\u8fd8\u9644\u4e0a\u4e86\u5173\u952e\u7684\u5b9e\u9a8c\u6ce8\u610f\u4e8b\u9879\u4e0e\u540e\u671f\u56fe\u50cf\u5904\u7406\u5efa\u8bae\u3002<\/p>\n","protected":false},"author":1,"featured_media":0,"comment_status":"open","ping_status":"closed","sticky":false,"template":"","format":"standard","meta":{"footnotes":""},"categories":[4],"tags":[],"class_list":["post-13539","post","type-post","status-publish","format-standard","hentry","category-note"],"_links":{"self":[{"href":"https:\/\/fanyiming.life\/index.php?rest_route=\/wp\/v2\/posts\/13539","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/fanyiming.life\/index.php?rest_route=\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/fanyiming.life\/index.php?rest_route=\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/fanyiming.life\/index.php?rest_route=\/wp\/v2\/users\/1"}],"replies":[{"embeddable":true,"href":"https:\/\/fanyiming.life\/index.php?rest_route=%2Fwp%2Fv2%2Fcomments&post=13539"}],"version-history":[{"count":6,"href":"https:\/\/fanyiming.life\/index.php?rest_route=\/wp\/v2\/posts\/13539\/revisions"}],"predecessor-version":[{"id":13737,"href":"https:\/\/fanyiming.life\/index.php?rest_route=\/wp\/v2\/posts\/13539\/revisions\/13737"}],"wp:attachment":[{"href":"https:\/\/fanyiming.life\/index.php?rest_route=%2Fwp%2Fv2%2Fmedia&parent=13539"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/fanyiming.life\/index.php?rest_route=%2Fwp%2Fv2%2Fcategories&post=13539"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/fanyiming.life\/index.php?rest_route=%2Fwp%2Fv2%2Ftags&post=13539"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}