{"id":625,"date":"2015-02-09T00:47:13","date_gmt":"2015-02-09T00:47:13","guid":{"rendered":"http:\/\/joekitz.com\/LungMap\/?p=625"},"modified":"2018-08-14T16:50:13","modified_gmt":"2018-08-14T16:50:13","slug":"e16-5_c57bl6_sox2_nkx2-1_acta2_confocal-imaging","status":"publish","type":"post","link":"https:\/\/research.cchmc.org\/lungimage\/?p=625","title":{"rendered":"E16.5 C57Bl6 SOX2, NKX2.1, and ACTA2 Confocal Imaging"},"content":{"rendered":"\t\t<div data-elementor-type=\"wp-post\" data-elementor-id=\"625\" class=\"elementor elementor-625 elementor-bc-flex-widget\" data-elementor-post-type=\"post\">\n\t\t\t\t\t\t<section class=\"elementor-section elementor-top-section elementor-element elementor-element-14962793 elementor-section-boxed elementor-section-height-default elementor-section-height-default\" data-id=\"14962793\" data-element_type=\"section\">\n\t\t\t\t\t\t<div class=\"elementor-container elementor-column-gap-default\">\n\t\t\t\t\t<div class=\"elementor-column elementor-col-50 elementor-top-column elementor-element elementor-element-3814a908\" data-id=\"3814a908\" data-element_type=\"column\">\n\t\t\t<div class=\"elementor-widget-wrap elementor-element-populated\">\n\t\t\t\t\t\t<div class=\"elementor-element elementor-element-619c47fd elementor-widget elementor-widget-theme-post-title elementor-page-title elementor-widget-heading\" data-id=\"619c47fd\" data-element_type=\"widget\" data-widget_type=\"theme-post-title.default\">\n\t\t\t\t<div class=\"elementor-widget-container\">\n\t\t\t\t\t<h1 class=\"elementor-heading-title elementor-size-large\">E16.5 C57Bl6 SOX2, NKX2.1, and ACTA2 Confocal Imaging<\/h1>\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t<section class=\"elementor-section elementor-inner-section elementor-element elementor-element-5668928d elementor-section-boxed elementor-section-height-default elementor-section-height-default\" data-id=\"5668928d\" data-element_type=\"section\">\n\t\t\t\t\t\t<div class=\"elementor-container elementor-column-gap-default\">\n\t\t\t\t\t<div class=\"elementor-column elementor-col-100 elementor-inner-column elementor-element elementor-element-30a39f11\" data-id=\"30a39f11\" data-element_type=\"column\">\n\t\t\t<div class=\"elementor-widget-wrap elementor-element-populated\">\n\t\t\t\t\t\t<div class=\"elementor-element elementor-element-2b1d84e5 elementor-widget elementor-widget-shortcode\" data-id=\"2b1d84e5\" data-element_type=\"widget\" data-widget_type=\"shortcode.default\">\n\t\t\t\t<div class=\"elementor-widget-container\">\n\t\t\t\t\t\t\t<div class=\"elementor-shortcode\"><div class=\"wonderplugingallery-container\" id=\"wonderplugingallery-container-3\" style=\"max-width:550px;margin:0 auto;\"><div class=\"wonderplugingallery\" id=\"wonderplugingallery-3\" data-galleryid=\"3\" data-width=\"550\" data-height=\"550\" data-skin=\"light\" data-autoslide=\"false\" data-autoplayvideo=\"false\" data-html5player=\"true\" data-responsive=\"true\" data-fullwidth=\"false\" data-showtitle=\"false\" data-showdescription=\"false\" data-showplaybutton=\"false\" data-showfullscreenbutton=\"true\" data-showtimer=\"false\" data-showcarousel=\"true\" data-galleryshadow=\"true\" data-slideshadow=\"true\" data-thumbshowtitle=\"false\" data-thumbshadow=\"true\" data-lightboxshowtitle=\"true\" data-lightboxshowdescription=\"true\" data-specifyid=\"true\" data-showimgtitle=\"false\" data-titlesmallscreen=\"false\" data-initsocial=\"false\" data-showsocial=\"false\" data-showemail=\"false\" data-showfacebook=\"false\" data-showtwitter=\"false\" data-showpinterest=\"false\" data-socialrotateeffect=\"false\" data-doshortcodeontext=\"false\" data-googleanalyticsaccount=\"\" data-resizemode=\"fit\" data-imagetoolboxmode=\"show\" data-effect=\"fade\" data-padding=\"12\" data-bgcolor=\"\" data-bgimage=\"\" data-thumbwidth=\"100\" data-thumbheight=\"100\" data-thumbgap=\"8\" data-thumbrowgap=\"16\" data-lightboxtextheight=\"72\" data-lightboxtitlecss=\"{color:#333333; font:bold 12px Arial,Helvetica,sans-serif; overflow:hidden; white-space:normal; text-align:Center; line-height:18px;}\" data-lightboxdescriptioncss=\"{color:#333333; font:normal 12px Arial,Helvetica,sans-serif; overflow:hidden; white-space:normal; text-align:Center; line-height:14px;}\" data-titlecss=\"{color:#ffffff; font-size:14px; font-family:Armata, sans-serif, Arial; overflow:hidden; white-space:normal; text-align:Center; padding:10px 0px 10px 10px;  background:rgb(102, 102, 102) transparent; background:rgba(102, 102, 102, 0.6);}\" data-descriptioncss=\"{color:#ffffff; font-size:12px; font-family:Armata, sans-serif, Arial; overflow:hidden; white-space:normal; text-align:Center; padding:0px 0px 10px 10px;  background:rgb(102, 102, 102) transparent; background:rgba(102, 102, 102, 0.6);}\" data-jsfolder=\"https:\/\/research.cchmc.org\/lungimage\/wp-content\/plugins\/wonderplugin-gallery\/engine\/\" style=\"display:none;\" ><a class=\"html5galleryimglink\" href=\"https:\/\/research.cchmc.org\/lungimage\/wp-content\/uploads\/2015\/02\/2014-012-019_C57Bl6_E16.5_LMM.14.24.8.7_SOX2-NKX2.1-ACTA2-01.jpg\" data-mediatype=1><img decoding=\"async\" class=\"lazyload html5galleryimg html5gallery-tn-image\" src=\"data:image\/svg+xml,%3Csvg%20xmlns%3D%27http%3A%2F%2Fwww.w3.org%2F2000%2Fsvg%27%20width%3D%271024%27%20height%3D%271024%27%20viewBox%3D%270%200%201024%201024%27%3E%3Crect%20width%3D%271024%27%20height%3D%271024%27%20fill-opacity%3D%220%22%2F%3E%3C%2Fsvg%3E\" data-orig-src=\"https:\/\/research.cchmc.org\/lungimage\/wp-content\/uploads\/2015\/02\/2014-012-019_C57Bl6_E16.5_LMM.14.24.8.7_SOX2-NKX2.1-ACTA2-01.jpg\" alt=\"E16.5 Immunofluorescence and Confocal Imaging Showing Protein Expression of SOX2, NKX2.1, and ACTA2\" data-description=\"20X Tile Scan\"><\/a><div class=\"html5gallery-info\"><div class=\"html5gallery-title\">E16.5 Immunofluorescence and Confocal Imaging Showing Protein Expression of SOX2, NKX2.1, and ACTA2<\/div><div class=\"html5gallery-description\">20X Tile Scan<\/div><\/div><a class=\"html5galleryimglink\" href=\"https:\/\/research.cchmc.org\/lungimage\/wp-content\/uploads\/2015\/02\/2014-012-019_C57Bl6_E16.5_LMM.14.24.8.7_SOX2-NKX2.1-ACTA2-02.jpg\" data-mediatype=1><img decoding=\"async\" class=\"lazyload html5galleryimg html5gallery-tn-image\" src=\"data:image\/svg+xml,%3Csvg%20xmlns%3D%27http%3A%2F%2Fwww.w3.org%2F2000%2Fsvg%27%20width%3D%271024%27%20height%3D%271024%27%20viewBox%3D%270%200%201024%201024%27%3E%3Crect%20width%3D%271024%27%20height%3D%271024%27%20fill-opacity%3D%220%22%2F%3E%3C%2Fsvg%3E\" data-orig-src=\"https:\/\/research.cchmc.org\/lungimage\/wp-content\/uploads\/2015\/02\/2014-012-019_C57Bl6_E16.5_LMM.14.24.8.7_SOX2-NKX2.1-ACTA2-02.jpg\" alt=\"E16.5 Immunofluorescence and Confocal Imaging Showing Protein Expression of SOX2, NKX2.1, and ACTA2\" data-description=\"20X\"><\/a><div class=\"html5gallery-info\"><div class=\"html5gallery-title\">E16.5 Immunofluorescence and Confocal Imaging Showing Protein Expression of SOX2, NKX2.1, and ACTA2<\/div><div class=\"html5gallery-description\">20X<\/div><\/div><a class=\"html5galleryimglink\" href=\"https:\/\/research.cchmc.org\/lungimage\/wp-content\/uploads\/2015\/02\/2014-012-019_C57Bl6_E16.5_LMM.14.24.8.7_SOX2-NKX2.1-ACTA2-03.jpg\" data-mediatype=1><img decoding=\"async\" class=\"lazyload html5galleryimg html5gallery-tn-image\" src=\"data:image\/svg+xml,%3Csvg%20xmlns%3D%27http%3A%2F%2Fwww.w3.org%2F2000%2Fsvg%27%20width%3D%271024%27%20height%3D%271024%27%20viewBox%3D%270%200%201024%201024%27%3E%3Crect%20width%3D%271024%27%20height%3D%271024%27%20fill-opacity%3D%220%22%2F%3E%3C%2Fsvg%3E\" data-orig-src=\"https:\/\/research.cchmc.org\/lungimage\/wp-content\/uploads\/2015\/02\/2014-012-019_C57Bl6_E16.5_LMM.14.24.8.7_SOX2-NKX2.1-ACTA2-03.jpg\" alt=\"E16.5 Immunofluorescence and Confocal Imaging Showing Protein Expression of SOX2, NKX2.1, and ACTA2\" data-description=\"60X\"><\/a><div class=\"html5gallery-info\"><div class=\"html5gallery-title\">E16.5 Immunofluorescence and Confocal Imaging Showing Protein Expression of SOX2, NKX2.1, and ACTA2<\/div><div class=\"html5gallery-description\">60X<\/div><\/div><a class=\"html5galleryimglink\" href=\"https:\/\/research.cchmc.org\/lungimage\/wp-content\/uploads\/2015\/02\/2014-012-019_C57Bl6_E16.5_LMM.14.24.8.7_SOX2-NKX2.1-ACTA2-04.jpg\" data-mediatype=1><img decoding=\"async\" class=\"lazyload html5galleryimg html5gallery-tn-image\" src=\"data:image\/svg+xml,%3Csvg%20xmlns%3D%27http%3A%2F%2Fwww.w3.org%2F2000%2Fsvg%27%20width%3D%271024%27%20height%3D%271024%27%20viewBox%3D%270%200%201024%201024%27%3E%3Crect%20width%3D%271024%27%20height%3D%271024%27%20fill-opacity%3D%220%22%2F%3E%3C%2Fsvg%3E\" data-orig-src=\"https:\/\/research.cchmc.org\/lungimage\/wp-content\/uploads\/2015\/02\/2014-012-019_C57Bl6_E16.5_LMM.14.24.8.7_SOX2-NKX2.1-ACTA2-04.jpg\" alt=\"E16.5 Immunofluorescence and Confocal Imaging Showing Protein Expression of SOX2, NKX2.1, and ACTA2\" data-description=\"20X\"><\/a><div class=\"html5gallery-info\"><div class=\"html5gallery-title\">E16.5 Immunofluorescence and Confocal Imaging Showing Protein Expression of SOX2, NKX2.1, and ACTA2<\/div><div class=\"html5gallery-description\">20X<\/div><\/div><a class=\"html5galleryimglink\" href=\"https:\/\/research.cchmc.org\/lungimage\/wp-content\/uploads\/2015\/02\/2014-012-019_C57Bl6_E16.5_LMM.14.24.8.7_SOX2-NKX2.1-ACTA2-05.jpg\" data-mediatype=1><img decoding=\"async\" class=\"lazyload html5galleryimg html5gallery-tn-image\" src=\"data:image\/svg+xml,%3Csvg%20xmlns%3D%27http%3A%2F%2Fwww.w3.org%2F2000%2Fsvg%27%20width%3D%271024%27%20height%3D%271024%27%20viewBox%3D%270%200%201024%201024%27%3E%3Crect%20width%3D%271024%27%20height%3D%271024%27%20fill-opacity%3D%220%22%2F%3E%3C%2Fsvg%3E\" data-orig-src=\"https:\/\/research.cchmc.org\/lungimage\/wp-content\/uploads\/2015\/02\/2014-012-019_C57Bl6_E16.5_LMM.14.24.8.7_SOX2-NKX2.1-ACTA2-05.jpg\" alt=\"E16.5 Immunofluorescence and Confocal Imaging Showing Protein Expression of SOX2, NKX2.1, and ACTA2\" data-description=\"60X\"><\/a><div class=\"html5gallery-info\"><div class=\"html5gallery-title\">E16.5 Immunofluorescence and Confocal Imaging Showing Protein Expression of SOX2, NKX2.1, and ACTA2<\/div><div class=\"html5gallery-description\">60X<\/div><\/div><a class=\"html5galleryimglink\" href=\"https:\/\/research.cchmc.org\/lungimage\/wp-content\/uploads\/2015\/02\/2014-012-019_C57Bl6_E16.5_LMM.14.24.8.7_SOX2-NKX2.1-ACTA2-06.jpg\" data-mediatype=1><img decoding=\"async\" class=\"lazyload html5galleryimg html5gallery-tn-image\" src=\"data:image\/svg+xml,%3Csvg%20xmlns%3D%27http%3A%2F%2Fwww.w3.org%2F2000%2Fsvg%27%20width%3D%271024%27%20height%3D%271024%27%20viewBox%3D%270%200%201024%201024%27%3E%3Crect%20width%3D%271024%27%20height%3D%271024%27%20fill-opacity%3D%220%22%2F%3E%3C%2Fsvg%3E\" data-orig-src=\"https:\/\/research.cchmc.org\/lungimage\/wp-content\/uploads\/2015\/02\/2014-012-019_C57Bl6_E16.5_LMM.14.24.8.7_SOX2-NKX2.1-ACTA2-06.jpg\" alt=\"E16.5 Immunofluorescence and Confocal Imaging Showing Protein Expression of SOX2, NKX2.1, and ACTA2\" data-description=\"20X\"><\/a><div class=\"html5gallery-info\"><div class=\"html5gallery-title\">E16.5 Immunofluorescence and Confocal Imaging Showing Protein Expression of SOX2, NKX2.1, and ACTA2<\/div><div class=\"html5gallery-description\">20X<\/div><\/div><a class=\"html5galleryimglink\" href=\"https:\/\/research.cchmc.org\/lungimage\/wp-content\/uploads\/2015\/02\/2014-012-019_C57Bl6_E16.5_LMM.14.24.8.7_SOX2-NKX2.1-ACTA2-07.jpg\" data-mediatype=1><img decoding=\"async\" class=\"lazyload html5galleryimg html5gallery-tn-image\" src=\"data:image\/svg+xml,%3Csvg%20xmlns%3D%27http%3A%2F%2Fwww.w3.org%2F2000%2Fsvg%27%20width%3D%271024%27%20height%3D%271024%27%20viewBox%3D%270%200%201024%201024%27%3E%3Crect%20width%3D%271024%27%20height%3D%271024%27%20fill-opacity%3D%220%22%2F%3E%3C%2Fsvg%3E\" data-orig-src=\"https:\/\/research.cchmc.org\/lungimage\/wp-content\/uploads\/2015\/02\/2014-012-019_C57Bl6_E16.5_LMM.14.24.8.7_SOX2-NKX2.1-ACTA2-07.jpg\" alt=\"E16.5 Immunofluorescence and Confocal Imaging Showing Protein Expression of SOX2, NKX2.1, and ACTA2\" data-description=\"60X\"><\/a><div class=\"html5gallery-info\"><div class=\"html5gallery-title\">E16.5 Immunofluorescence and Confocal Imaging Showing Protein Expression of SOX2, NKX2.1, and ACTA2<\/div><div class=\"html5gallery-description\">60X<\/div><\/div><\/div><\/div><\/div>\n\t\t\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t<div class=\"elementor-element elementor-element-0f18709 elementor-widget elementor-widget-template\" data-id=\"0f18709\" data-element_type=\"widget\" data-widget_type=\"template.default\">\n\t\t\t\t<div class=\"elementor-widget-container\">\n\t\t\t\t\t\t\t<div class=\"elementor-template\">\n\t\t\t\t\t<div data-elementor-type=\"page\" data-elementor-id=\"24332\" class=\"elementor elementor-24332 elementor-bc-flex-widget\" data-elementor-post-type=\"elementor_library\">\n\t\t\t\t\t\t<section class=\"elementor-section elementor-top-section elementor-element elementor-element-4650a05 elementor-section-boxed elementor-section-height-default elementor-section-height-default\" data-id=\"4650a05\" data-element_type=\"section\">\n\t\t\t\t\t\t<div class=\"elementor-container elementor-column-gap-default\">\n\t\t\t\t\t<div class=\"elementor-column elementor-col-50 elementor-top-column elementor-element elementor-element-951322a\" data-id=\"951322a\" data-element_type=\"column\">\n\t\t\t<div class=\"elementor-widget-wrap\">\n\t\t\t\t\t\t\t<\/div>\n\t\t<\/div>\n\t\t\t\t<div class=\"elementor-column elementor-col-50 elementor-top-column elementor-element elementor-element-4f951ba\" data-id=\"4f951ba\" data-element_type=\"column\">\n\t\t\t<div class=\"elementor-widget-wrap\">\n\t\t\t\t\t\t\t<\/div>\n\t\t<\/div>\n\t\t\t\t\t<\/div>\n\t\t<\/section>\n\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t\t<\/div>\n\t\t<\/div>\n\t\t\t\t\t<\/div>\n\t\t<\/section>\n\t\t\t\t<section class=\"elementor-section elementor-inner-section elementor-element elementor-element-52315a42 elementor-section-boxed elementor-section-height-default elementor-section-height-default\" data-id=\"52315a42\" data-element_type=\"section\">\n\t\t\t\t\t\t<div class=\"elementor-container elementor-column-gap-default\">\n\t\t\t\t\t<div class=\"elementor-column elementor-col-100 elementor-inner-column elementor-element elementor-element-64541643\" data-id=\"64541643\" data-element_type=\"column\">\n\t\t\t<div class=\"elementor-widget-wrap elementor-element-populated\">\n\t\t\t\t\t\t<div class=\"elementor-element elementor-element-50a02117 elementor-widget elementor-widget-text-editor\" data-id=\"50a02117\" data-element_type=\"widget\" data-widget_type=\"text-editor.default\">\n\t\t\t\t<div class=\"elementor-widget-container\">\n\t\t\t\t\t\t\t\t\t<p style=\"text-align: center;\"><span style=\"font-size: 14pt;\"><strong>Immunofluorescence- SOX2<\/strong>,<strong> NKX2.1, <\/strong>and <strong><a href=\"http:\/\/www.sigmaaldrich.com\/catalog\/product\/sigma\/a5228?lang=en&amp;region=US\" target=\"_blank\" rel=\"noopener\">ACTA2<\/a><\/strong><\/span><strong style=\"letter-spacing: 0px;\">\u00a0<\/strong><\/p><p><strong>Purpose:\u00a0 <\/strong>Immunofluorescence on slides of 7\u00b5m frozen sections of C57BL6 E16.5 lungs for<strong> SOX2<\/strong>, <strong>NKX2.1, <\/strong>and <a href=\"http:\/\/www.sigmaaldrich.com\/catalog\/product\/sigma\/a5228?lang=en&amp;region=US\" target=\"_blank\" rel=\"noopener\"><strong>ACTA2<\/strong><\/a> with antigen retrieval.<\/p><p><strong>Day 1<\/strong><\/p><p>1. Paraffin sections are placed at 60<sup>o<\/sup>C for 2 hours to overnight to melt paraffin.<br \/>2. Paraffin sections are then placed in xylene 3X for 10 minutes each, followed by 3X in 100% ETOH for 3 minutes each, 95% ETOH for 3 minutes each, and 70% ETOH for 3 minutes each. Slides are then placed in 1X PBS for 5 minutes to completely rehydrate tissue.<br \/>3. Briefly equilibrate slides in Antigen Retrieval Buffer.<br \/>4. 10 mM sodium citrate, pH 6.0, and heat in a microwave at 96<sup>o<\/sup>C.\u00a0*We usually do a series of three runs (6-7 minutes each run) to equal the time\/temp because of evaporation (refill coplin jars with dH<sub>2<\/sub>O).<br \/>5. Microwave according to instructions on microwave.<br \/>6. Cool on countertop, 15 min.<br \/>7. Rinse with dH2O<br \/>8. 1X PBS, 5 min.<br \/>9. Block in 4% Goat serum\/PBS-T, 2 hours at RT.<br \/>10. For Rabbit anit-SOX2 (R1236, BL 5\/7\/09, Seven Hills) dilute 1:200, For guinea pig anti-NKX2.1 (G237, Bl. 10\/7\/11) dilute 1:500, for mouse anti-<strong><a href=\"http:\/\/www.sigmaaldrich.com\/catalog\/product\/sigma\/a5228?lang=en\u00aeion=US\" target=\"_blank\" rel=\"noopener\"> ACTA2<\/a><\/strong> (<strong><a href=\"http:\/\/www.sigmaaldrich.com\/catalog\/product\/sigma\/a5228?lang=en\u00aeion=US\" target=\"_blank\" rel=\"noopener\">A5228<\/a><\/strong>, Sigma) dilute 1:2000 in blocking buffer and incubate on tissue overnight @ 4oC.<\/p><p><strong>Day 2<\/strong><\/p><p>1. Rinse slides in PBS-T 3X, 5 min.<br \/>2. Apply secondary antibody, Goat Alexa Fluor 488 anti-rabbit IgG (<strong><a href=\"https:\/\/www.lifetechnologies.com\/order\/genome-database\/antibody\/Rabbit-IgG-H-L-Secondary-Antibody-Polyclonal\/A-11034\" target=\"_blank\" rel=\"noopener\">A11034<\/a><\/strong>, Lot# 1298480, for anti-<strong>SOX2<\/strong>), Goat Alexa Fluor 555 anti-guinea pig IgG (<strong><a href=\"https:\/\/www.lifetechnologies.com\/order\/genome-database\/antibody\/Guinea-Pig-IgG-H-L-Secondary-Antibody-Polyclonal\/A-21435\" target=\"_blank\" rel=\"noopener\">A21435<\/a><\/strong>, Lot# 1571699, for anti-NKX2.1) at 1:200 at 1:200, Donkey Alexa Fluor 647 anti-mouse IgG (<strong><a href=\"https:\/\/www.lifetechnologies.com\/order\/genome-database\/antibody\/Mouse-IgG-H-L-Secondary-Antibody-Polyclonal\/A-31571\" target=\"_blank\" rel=\"noopener\">A31571<\/a><\/strong>, Lot# 1366514, for anti-<strong><a href=\"http:\/\/www.sigmaaldrich.com\/catalog\/product\/sigma\/a5228?lang=en\u00aeion=US\" target=\"_blank\" rel=\"noopener\">ACTA2<\/a><\/strong> ) at 1:200, in blocking buffer. Spin down antibody cocktail in a \u00b5fuge for 10 min and incubated at room temperature for 1 hour.<br \/>3. Rinse in PBS-T 3X, 5 min.<br \/>4. Dilute DAPI 1:2000 and apply to slides for 10 min.<br \/>5. Wash in PBS-T 3X, 5 min.<br \/>6. Rinse slides in 0.1M PB or TB, 3X, 5 min.<br \/>7. Add 1 drop of Prolong Gold anti-fade mounting medium (P36930).<br \/>8. Coverslip with Gold Seal Coverslip (Cat# 3422 Electron Microscopy Sciences, 22 X 22 mm).<\/p><p>9. Allow Prolong Gold to cure overnight at room temp in light sealed box.<\/p><p><strong>Tissue Used:<\/strong><\/p><p>LMM.14.24.8.7 E16.5<\/p><p><span style=\"letter-spacing: 0px;\">Gender: Male<\/span><\/p><p>Crown-to-Rump Length: 16.5 mm<\/p><p>Weight=0.644g<\/p><p><strong>Appendix<\/strong><\/p><p><strong>Antigen Retrieval Solution<\/strong><br \/>18ml Solution A- 0.1M Citric Acid (Sigma, C1909) pH 2.5<br \/>82ml Reagent B- 0.1M Sodium Citrate (Sigma, S4641 ) pH 8.2<br \/>1L dH2O<br \/>pH 6.0<\/p>\t\t\t\t\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t\t<\/div>\n\t\t<\/div>\n\t\t\t\t\t<\/div>\n\t\t<\/section>\n\t\t\t\t<section class=\"elementor-section elementor-inner-section elementor-element elementor-element-43cf34f7 elementor-section-boxed elementor-section-height-default elementor-section-height-default\" data-id=\"43cf34f7\" data-element_type=\"section\">\n\t\t\t\t\t\t<div class=\"elementor-container elementor-column-gap-default\">\n\t\t\t\t\t<div class=\"elementor-column elementor-col-100 elementor-inner-column elementor-element elementor-element-73c1e99e\" data-id=\"73c1e99e\" data-element_type=\"column\">\n\t\t\t<div class=\"elementor-widget-wrap elementor-element-populated\">\n\t\t\t\t\t\t<div class=\"elementor-element elementor-element-11d9635c elementor-widget elementor-widget-facebook-comments\" data-id=\"11d9635c\" data-element_type=\"widget\" data-widget_type=\"facebook-comments.default\">\n\t\t\t\t<div class=\"elementor-widget-container\">\n\t\t\t\t\t\t\t<div class=\"elementor-facebook-widget fb-comments\" data-href=\"https:\/\/research.cchmc.org\/lungimage?p=625\" data-width=\"100%\" data-numposts=\"10\" data-order-by=\"social\" style=\"min-height: 1px\"><\/div>\n\t\t\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t\t<\/div>\n\t\t<\/div>\n\t\t\t\t\t<\/div>\n\t\t<\/section>\n\t\t\t\t\t<\/div>\n\t\t<\/div>\n\t\t\t\t<div class=\"elementor-column elementor-col-50 elementor-top-column elementor-element elementor-element-1893b04\" data-id=\"1893b04\" data-element_type=\"column\">\n\t\t\t<div class=\"elementor-widget-wrap elementor-element-populated\">\n\t\t\t\t\t\t<div class=\"elementor-element elementor-element-51cce891 elementor-widget elementor-widget-sidebar\" data-id=\"51cce891\" data-element_type=\"widget\" data-widget_type=\"sidebar.default\">\n\t\t\t\t<div class=\"elementor-widget-container\">\n\t\t\t\t\t<div id=\"search-8\" class=\"widget widget_search\">\t\t<form role=\"search\" class=\"searchform fusion-search-form  fusion-search-form-classic\" method=\"get\" action=\"https:\/\/research.cchmc.org\/lungimage\/\">\n\t\t\t<div class=\"fusion-search-form-content\">\n\n\t\t\t\t\n\t\t\t\t<div class=\"fusion-search-field search-field\">\n\t\t\t\t\t<label><span class=\"screen-reader-text\">Search for:<\/span>\n\t\t\t\t\t\t\t\t\t\t\t\t\t<input type=\"search\" value=\"\" name=\"s\" class=\"s\" placeholder=\"Search...\" required aria-required=\"true\" aria-label=\"Search...\"\/>\n\t\t\t\t\t\t\t\t\t\t\t<\/label>\n\t\t\t\t<\/div>\n\t\t\t\t<div class=\"fusion-search-button search-button\">\n\t\t\t\t\t<input type=\"submit\" class=\"fusion-search-submit searchsubmit\" aria-label=\"Search\" value=\"&#xf002;\" \/>\n\t\t\t\t\t\t\t\t\t<\/div>\n\n\t\t\t\t\n\t\t\t<\/div>\n\n\n\t\t\t\n\t\t<\/form>\n\t\t<\/div><div id=\"text-13\" class=\"widget widget_text\"><div class=\"heading\"><h4 class=\"widget-title\">Related Experiments<\/h4><\/div>\t\t\t<div class=\"textwidget\"><div class=\"wondercarousellightbox_options\" data-skinsfoldername=\"\"  data-jsfolder=\"https:\/\/research.cchmc.org\/lungimage\/wp-content\/plugins\/wonderplugin-carousel\/engine\/\" style=\"display:none;\"><\/div>\r\n<div class=\"wonderplugincarousel-container\" id=\"wonderplugincarousel-container-47\"><div class=\"wonderplugincarousel\" id=\"wonderplugincarousel-47\" data-carouselid=\"47\" data-width=\"250\" data-height=\"250\" data-skin=\"vertical\" 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data-lightboxbarheight=\"64\" data-lightboxtitlebottomcss=\"{color:#002e9b; font-size:14px; font-family:Armata,sans-serif,Arial; overflow:hidden; text-align:center;}\" data-lightboxdescriptionbottomcss=\"{color:#333; font-size:12px; font-family:Arial,Helvetica,sans-serif; overflow:hidden; text-align:left; margin:4px 0px 0px; padding: 0px;}\" data-continuousduration=\"2500\" data-autoplaydir=\"left\" data-scrollmode=\"page\" data-interval=\"3000\" data-transitionduration=\"1000\" data-lightboxtitlestyle=\"bottom\" data-lightboximagepercentage=\"75\" data-lightboxdefaultvideovolume=\"1\" data-lightboxoverlaybgcolor=\"#000\" data-lightboxoverlayopacity=\"0.9\" data-lightboxbgcolor=\"#fff\" data-lightboxtitleprefix=\"%NUM \/ %TOTAL\" data-lightboxtitleinsidecss=\"color:#fff; font-size:16px; font-family:Arial,Helvetica,sans-serif; overflow:hidden; text-align:left;\" data-lightboxdescriptioninsidecss=\"color:#fff; font-size:12px; font-family:Arial,Helvetica,sans-serif; overflow:hidden; 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data-jsfolder=\"https:\/\/research.cchmc.org\/lungimage\/wp-content\/plugins\/wonderplugin-carousel\/engine\/\" style=\"display:none;position:relative;margin:0 auto;width:100%;max-width:250px;\" ><div class=\"amazingcarousel-list-container\" style=\"overflow:hidden;\"><ul class=\"amazingcarousel-list\"><li class=\"amazingcarousel-item\"><div class=\"amazingcarousel-item-container\"><div class=\"amazingcarousel-image\">\n\t<a href=\"https:\/\/research.cchmc.org\/lungimage\/?p=625\" target=\"target=\"_blank\"\"><img decoding=\"async\" class=\"lazyload amazingcarousel-image-img\" src=\"data:image\/svg+xml,%3Csvg%20xmlns%3D%27http%3A%2F%2Fwww.w3.org%2F2000%2Fsvg%27%20width%3D%27450%27%20height%3D%27450%27%20viewBox%3D%270%200%20450%20450%27%3E%3Crect%20width%3D%27450%27%20height%3D%27450%27%20fill-opacity%3D%220%22%2F%3E%3C%2Fsvg%3E\" data-orig-src=\"https:\/\/research.cchmc.org\/lungimage\/wp-content\/uploads\/2015\/02\/2014-012-019_C57Bl6_E16.5_LMM.14.24.8.7_SOX2-NKX2.1-ACTA2-041.jpg\" alt=\"E16.5 C57BL6 SOX2, NKX2.1, and ACTA2 Confocal Imaging\" data-description=\"\" \/><\/a>\n\t<div class=\"amazingcarousel-text\">\n\t\t<div class=\"amazingcarousel-text-bg\"><\/div>\n\t\t<div class=\"amazingcarousel-title\">E16.5 C57BL6 SOX2, NKX2.1, and ACTA2 Confocal Imaging<\/div>\n\t<\/div>\n<\/div><\/div><\/li><li class=\"amazingcarousel-item\"><div class=\"amazingcarousel-item-container\"><div class=\"amazingcarousel-image\">\n\t<a href=\"https:\/\/research.cchmc.org\/lungimage\/?p=2008\" target=\"target=\"_blank\"\"><img decoding=\"async\" class=\"lazyload amazingcarousel-image-img\" src=\"data:image\/svg+xml,%3Csvg%20xmlns%3D%27http%3A%2F%2Fwww.w3.org%2F2000%2Fsvg%27%20width%3D%27250%27%20height%3D%27250%27%20viewBox%3D%270%200%20250%20250%27%3E%3Crect%20width%3D%27250%27%20height%3D%27250%27%20fill-opacity%3D%220%22%2F%3E%3C%2Fsvg%3E\" data-orig-src=\"https:\/\/research.cchmc.org\/lungimage\/wp-content\/uploads\/2015\/04\/2014-012-015_C57Bl6_E18.5_LMM.14.49.4.9_Sox2_NKX2.1_ACTA2-05_250X250.jpg\" alt=\"E18.5 C57BL6 SOX2, NKX2.1, and ACTA2 Confocal Imaging\" data-description=\"\" \/><\/a>\n\t<div class=\"amazingcarousel-text\">\n\t\t<div class=\"amazingcarousel-text-bg\"><\/div>\n\t\t<div class=\"amazingcarousel-title\">E18.5 C57BL6 SOX2, NKX2.1, and ACTA2 Confocal Imaging<\/div>\n\t<\/div>\n<\/div><\/div><\/li><li class=\"amazingcarousel-item\"><div class=\"amazingcarousel-item-container\"><div class=\"amazingcarousel-image\">\n\t<a href=\"https:\/\/research.cchmc.org\/lungimage\/?p=12796\" target=\"_blank\"><img decoding=\"async\" class=\"lazyload amazingcarousel-image-img\" src=\"data:image\/svg+xml,%3Csvg%20xmlns%3D%27http%3A%2F%2Fwww.w3.org%2F2000%2Fsvg%27%20width%3D%27300%27%20height%3D%27300%27%20viewBox%3D%270%200%20300%20300%27%3E%3Crect%20width%3D%27300%27%20height%3D%27300%27%20fill-opacity%3D%220%22%2F%3E%3C%2Fsvg%3E\" data-orig-src=\"https:\/\/research.cchmc.org\/lungimage\/wp-content\/uploads\/2017\/03\/2016-09-22_C57BL6_PND03_LMM.16.81A.3.57_SOX2_NKX2.1_ACTA2-05-1.jpg\" alt=\"Postnatal Day 3 C57BL6 SOX2, NKX2.1, and ACTA2 Confocal Imaging\" data-description=\"\" \/><\/a>\n\t<div class=\"amazingcarousel-text\">\n\t\t<div class=\"amazingcarousel-text-bg\"><\/div>\n\t\t<div class=\"amazingcarousel-title\">Postnatal Day 3 C57BL6 SOX2, NKX2.1, and ACTA2 Confocal Imaging<\/div>\n\t<\/div>\n<\/div><\/div><\/li><li class=\"amazingcarousel-item\"><div class=\"amazingcarousel-item-container\"><div class=\"amazingcarousel-image\">\n\t<a href=\"https:\/\/research.cchmc.org\/lungimage\/?p=2198\" target=\"target=\"_blank\"\"><img decoding=\"async\" class=\"lazyload amazingcarousel-image-img\" src=\"data:image\/svg+xml,%3Csvg%20xmlns%3D%27http%3A%2F%2Fwww.w3.org%2F2000%2Fsvg%27%20width%3D%27300%27%20height%3D%27300%27%20viewBox%3D%270%200%20300%20300%27%3E%3Crect%20width%3D%27300%27%20height%3D%27300%27%20fill-opacity%3D%220%22%2F%3E%3C%2Fsvg%3E\" data-orig-src=\"https:\/\/research.cchmc.org\/lungimage\/wp-content\/uploads\/2015\/04\/2015-003-030_C57Bl6_PND7_LMM.15.17A..5.12_SOX2_NKX21_ACTA2-05_300X300.jpg\" alt=\"Postnatal Day 7 C57BL6 SOX2, NKX2.1, and ACTA2 Confocal Imaging\" data-description=\"\" \/><\/a>\n\t<div class=\"amazingcarousel-text\">\n\t\t<div class=\"amazingcarousel-text-bg\"><\/div>\n\t\t<div class=\"amazingcarousel-title\">Postnatal Day 7 C57BL6 SOX2, NKX2.1, and ACTA2 Confocal Imaging<\/div>\n\t<\/div>\n<\/div><\/div><\/li><li class=\"amazingcarousel-item\"><div class=\"amazingcarousel-item-container\"><div class=\"amazingcarousel-image\">\n\t<a href=\"https:\/\/research.cchmc.org\/lungimage\/?p=12821\" target=\"_blank\"><img decoding=\"async\" class=\"lazyload amazingcarousel-image-img\" src=\"data:image\/svg+xml,%3Csvg%20xmlns%3D%27http%3A%2F%2Fwww.w3.org%2F2000%2Fsvg%27%20width%3D%27300%27%20height%3D%27300%27%20viewBox%3D%270%200%20300%20300%27%3E%3Crect%20width%3D%27300%27%20height%3D%27300%27%20fill-opacity%3D%220%22%2F%3E%3C%2Fsvg%3E\" data-orig-src=\"https:\/\/research.cchmc.org\/lungimage\/wp-content\/uploads\/2017\/03\/2016-09-27_C57BL6_PND10_LMM.16.92A.6L.55_SOX2_NKX2.1_ACTA2-10-1.jpg\" alt=\"Postnatal Day 10 C57BL6 SOX2, NKX2.1, and ACTA2 Confocal Imaging\" data-description=\"\" \/><\/a>\n\t<div class=\"amazingcarousel-text\">\n\t\t<div class=\"amazingcarousel-text-bg\"><\/div>\n\t\t<div class=\"amazingcarousel-title\">Postnatal Day 10 C57BL6 SOX2, NKX2.1, and ACTA2 Confocal Imaging<\/div>\n\t<\/div>\n<\/div><\/div><\/li><li class=\"amazingcarousel-item\"><div class=\"amazingcarousel-item-container\"><div class=\"amazingcarousel-image\">\n\t<a href=\"https:\/\/research.cchmc.org\/lungimage\/?p=6603\" target=\"target=\"_Blank\"\"><img decoding=\"async\" class=\"lazyload amazingcarousel-image-img\" src=\"data:image\/svg+xml,%3Csvg%20xmlns%3D%27http%3A%2F%2Fwww.w3.org%2F2000%2Fsvg%27%20width%3D%27300%27%20height%3D%27300%27%20viewBox%3D%270%200%20300%20300%27%3E%3Crect%20width%3D%27300%27%20height%3D%27300%27%20fill-opacity%3D%220%22%2F%3E%3C%2Fsvg%3E\" data-orig-src=\"https:\/\/research.cchmc.org\/lungimage\/wp-content\/uploads\/2016\/04\/2016-03-01_C57BL6_PND14_LMM.15.33A.6L.56_SOX2_NKX2.1_ACTA2-09-1.jpg\" alt=\"Postnatal Day 14 C57BL6 SOX2, NKX2.1, and ACTA2 Confocal Imaging\" data-description=\"\" \/><\/a>\n\t<div class=\"amazingcarousel-text\">\n\t\t<div class=\"amazingcarousel-text-bg\"><\/div>\n\t\t<div class=\"amazingcarousel-title\">Postnatal Day 14 C57BL6 SOX2, NKX2.1, and ACTA2 Confocal Imaging<\/div>\n\t<\/div>\n<\/div><\/div><\/li><li class=\"amazingcarousel-item\"><div class=\"amazingcarousel-item-container\"><div class=\"amazingcarousel-image\">\n\t<a href=\"https:\/\/research.cchmc.org\/lungimage\/?p=2200\" target=\"target=\"_Blank\"\"><img decoding=\"async\" class=\"lazyload amazingcarousel-image-img\" src=\"data:image\/svg+xml,%3Csvg%20xmlns%3D%27http%3A%2F%2Fwww.w3.org%2F2000%2Fsvg%27%20width%3D%27300%27%20height%3D%27300%27%20viewBox%3D%270%200%20300%20300%27%3E%3Crect%20width%3D%27300%27%20height%3D%27300%27%20fill-opacity%3D%220%22%2F%3E%3C%2Fsvg%3E\" data-orig-src=\"https:\/\/research.cchmc.org\/lungimage\/wp-content\/uploads\/2015\/04\/2015-004-006_C57Bl6_PND28_LMM.15.20A..1.30_SOX2_NKX21_ACTA2-03_300X300.jpg\" alt=\"Postnatal Day 28 C57BL6 SOX2, NKX2.1, and ACTA2 Confocal Imaging\" data-description=\"\" \/><\/a>\n\t<div class=\"amazingcarousel-text\">\n\t\t<div class=\"amazingcarousel-text-bg\"><\/div>\n\t\t<div class=\"amazingcarousel-title\">Postnatal Day 28 C57BL6 SOX2, NKX2.1, and ACTA2 Confocal Imaging<\/div>\n\t<\/div>\n<\/div><\/div><\/li><\/ul><div class=\"amazingcarousel-prev\"><\/div><div class=\"amazingcarousel-next\"><\/div><\/div><div class=\"amazingcarousel-nav\"><\/div><\/div><\/div><script>function wondercarousel_47_appendcss(csscode) {var head=document.head || document.getElementsByTagName(\"head\")[0];var style=document.createElement(\"style\");head.appendChild(style);style.type=\"text\/css\";if (style.styleSheet){style.styleSheet.cssText=csscode;} else {style.appendChild(document.createTextNode(csscode));}};wondercarousel_47_appendcss(\"@import url(https:\/\/fonts.googleapis.com\/css?family=Open+Sans);  #wonderplugincarousel-47 .amazingcarousel-image {\t \tposition: relative; \tpadding: 0px; }  #wonderplugincarousel-47 .amazingcarousel-image img { \tdisplay: block; \twidth: 100%; \tmax-width: 100%; \tborder: 0; \tmargin: 0; \tpadding: 0; \t-moz-border-radius: 0px; \t-webkit-border-radius: 0px; \tborder-radius: 0px; \t-moz-box-shadow:  0 1px 4px rgba(0, 0, 0, 0.2); \t-webkit-box-shadow: 0 1px 4px rgba(0, 0, 0, 0.2); \tbox-shadow: 0 1px 4px rgba(0, 0, 0, 0.2); }  #wonderplugincarousel-47 .amazingcarousel-text { \tposition:absolute; \tleft: 0px; \tbottom: 0px; \ttext-align:center; \twidth: 100%; \tdisplay: none; }  #wonderplugincarousel-47 .amazingcarousel-image:hover div.amazingcarousel-text {\t \tdisplay: block; }  #wonderplugincarousel-47 .amazingcarousel-text:hover { \tdisplay: block; }  #wonderplugincarousel-47 .amazingcarousel-text-bg { \tposition:absolute; \ttop: 0px; \tleft: 0px; \twidth: 100%; \theight: 100%; \tbackground-color: #333333; \topacity: 0.5; \tfilter: alpha(opacity=50); }  #wonderplugincarousel-47 .amazingcarousel-title { \tposition:relative; \tfont:14px \\\"Open Sans\\\", sans-serif; \tcolor:#ffffff; \ttext-align:center; \twidth: 100%; \tpadding: 10px 0px; }  #wonderplugincarousel-47 .amazingcarousel-list-container {  \tpadding: 0; }  #wonderplugincarousel-47 .amazingcarousel-item-container { \ttext-align: center; \tpadding: 8px; \tbackground-color: #fff; \tborder: 1px solid 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ease-in; } .amazingcarousel-socialmedia-rotate:hover { transform: rotate(360deg); }\");<\/script><\/div>\n\t\t<\/div>\n\t\t<div id=\"recent-posts-7\" class=\"widget widget_recent_entries\">\n\t\t<div class=\"heading\"><h4 class=\"widget-title\">Recent Experiments<\/h4><\/div>\n\t\t<ul>\n\t\t\t\t\t\t\t\t\t\t\t<li>\n\t\t\t\t\t<a href=\"https:\/\/research.cchmc.org\/lungimage\/?p=32066\">4 Year-Old Human Lung KRT14, MUC5B, and TUBA4A Confocal Imaging<\/a>\n\t\t\t\t\t\t\t\t\t<\/li>\n\t\t\t\t\t\t\t\t\t\t\t<li>\n\t\t\t\t\t<a href=\"https:\/\/research.cchmc.org\/lungimage\/?p=31223\">Alcian Blue Staining from PBR 3653 Patient with Cystic Fibrosis<\/a>\n\t\t\t\t\t\t\t\t\t<\/li>\n\t\t\t\t\t\t\t\t\t\t\t<li>\n\t\t\t\t\t<a href=\"https:\/\/research.cchmc.org\/lungimage\/?p=31202\">H&#038;E Staining from PBR 3383 Patient with Cystic Fibrosis<\/a>\n\t\t\t\t\t\t\t\t\t<\/li>\n\t\t\t\t\t\t\t\t\t\t\t<li>\n\t\t\t\t\t<a href=\"https:\/\/research.cchmc.org\/lungimage\/?p=31199\">H&#038;E Staining from PBR 3653 Patient with Cystic Fibrosis<\/a>\n\t\t\t\t\t\t\t\t\t<\/li>\n\t\t\t\t\t\t\t\t\t\t\t<li>\n\t\t\t\t\t<a href=\"https:\/\/research.cchmc.org\/lungimage\/?p=31196\">H&#038;E Staining of 31 Year-Old Human Donor 10<\/a>\n\t\t\t\t\t\t\t\t\t<\/li>\n\t\t\t\t\t\t\t\t\t\t\t<li>\n\t\t\t\t\t<a href=\"https:\/\/research.cchmc.org\/lungimage\/?p=31193\">H&#038;E Staining of 27 Year-Old Human Donor 3<\/a>\n\t\t\t\t\t\t\t\t\t<\/li>\n\t\t\t\t\t\t\t\t\t\t\t<li>\n\t\t\t\t\t<a href=\"https:\/\/research.cchmc.org\/lungimage\/?p=31190\">Alcian Blue Staining of 27 Year-Old Human Donor 3<\/a>\n\t\t\t\t\t\t\t\t\t<\/li>\n\t\t\t\t\t\t\t\t\t\t\t<li>\n\t\t\t\t\t<a href=\"https:\/\/research.cchmc.org\/lungimage\/?p=31186\">Alcian Blue Staining of 31 Year-Old Human Donor 10<\/a>\n\t\t\t\t\t\t\t\t\t<\/li>\n\t\t\t\t\t\t\t\t\t\t\t<li>\n\t\t\t\t\t<a href=\"https:\/\/research.cchmc.org\/lungimage\/?p=31182\">Alcian Blue Staining from PBR 3383 Patient with Cystic 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D071-17C2.20<\/a>\n\t\t\t\t\t\t\t\t\t<\/li>\n\t\t\t\t\t\t\t\t\t\t\t<li>\n\t\t\t\t\t<a href=\"https:\/\/research.cchmc.org\/lungimage\/?p=31007\">Alcian Blue Staining of 24 Year-Old Human Donor D071-16C3.27<\/a>\n\t\t\t\t\t\t\t\t\t<\/li>\n\t\t\t\t\t<\/ul>\n\n\t\t<\/div>\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t\t<\/div>\n\t\t<\/div>\n\t\t\t\t\t<\/div>\n\t\t<\/section>\n\t\t\t\t<\/div>\n\t\t","protected":false},"excerpt":{"rendered":"<p>Immunofluorescence- SOX2, NKX2.1, and ACTA2\u00a0 Purpose:\u00a0 Immunofluorescence on slides of 7\u00b5m frozen sections of C57BL6 E16.5 lungs for SOX2, NKX2.1, and ACTA2 with antigen retrieval. Day 1 1. Paraffin sections are placed at 60oC for 2 hours to overnight to melt paraffin.2. 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