{"id":19433,"date":"2018-02-23T17:06:26","date_gmt":"2018-02-23T17:06:26","guid":{"rendered":"https:\/\/research.cchmc.org\/lungimage\/?p=19433"},"modified":"2018-08-14T19:25:38","modified_gmt":"2018-08-14T19:25:38","slug":"postnatal-day-28-mouse-lung-second-harmonic-imaging","status":"publish","type":"post","link":"https:\/\/research.cchmc.org\/lungimage\/?p=19433","title":{"rendered":"Postnatal Day 28 Mouse Lung Second Harmonic Imaging"},"content":{"rendered":"<div class=\"fusion-fullwidth fullwidth-box fusion-builder-row-1 hundred-percent-fullwidth non-hundred-percent-height-scrolling\" style=\"--awb-border-radius-top-left:0px;--awb-border-radius-top-right:0px;--awb-border-radius-bottom-right:0px;--awb-border-radius-bottom-left:0px;--awb-overflow:visible;--awb-flex-wrap:wrap;\" ><div class=\"fusion-builder-row fusion-row\"><div class=\"fusion-layout-column fusion_builder_column fusion-builder-column-0 fusion_builder_column_1_1 1_1 fusion-one-full fusion-column-first fusion-column-last\" style=\"--awb-bg-size:cover;--awb-margin-bottom:25px;\"><div class=\"fusion-column-wrapper fusion-flex-column-wrapper-legacy\"><div class=\"fusion-text fusion-text-1\"><\/div><div class=\"fusion-text fusion-text-2\"><div class=\"wonderplugingallery-container\" id=\"wonderplugingallery-container-682\" style=\"max-width:550px;margin:0 auto;\"><div class=\"wonderplugingallery\" id=\"wonderplugingallery-682\" data-galleryid=\"682\" data-width=\"550\" data-height=\"550\" data-skin=\"light\" data-random=\"false\" data-autoslide=\"false\" data-autoplayvideo=\"false\" data-schemamarkup=\"false\" data-stopallplaying=\"false\" data-reloadonvideoend=\"false\" data-enabletabindex=\"false\" data-loadnextonvideoend=\"false\" data-hidetitlewhenvideoisplaying=\"false\" data-disablehovereventontouch=\"false\" data-autoslideandplayafterfirstplayed=\"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-donotinit=\"false\" data-addinitscript=\"false\" data-triggerresize=\"false\" data-thumbcolumnsresponsive=\"false\" data-showimgtitle=\"false\" data-titlesmallscreen=\"false\" data-initsocial=\"true\" data-showsocial=\"false\" data-showemail=\"false\" data-showfacebook=\"true\" data-showtwitter=\"true\" data-showpinterest=\"true\" data-socialrotateeffect=\"true\" data-doshortcodeontext=\"false\" data-duration=\"1500\" data-slideduration=\"1000\" data-slideshowinterval=\"6000\" 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-titleheight=\"72\" data-titlesmallscreenwidth=\"640\" data-titleheightsmallscreen=\"148\" data-socialmode=\"mouseover\" data-socialposition=\"position:absolute;top:8px;right:8px;\" data-socialpositionlightbox=\"position:absolute;top:8px;right:8px;\" data-socialdirection=\"horizontal\" data-socialbuttonsize=\"32\" data-socialbuttonfontsize=\"18\" data-triggerresizedelay=\"100\" data-thumbmediumsize=\"800\" data-thumbsmallsize=\"480\" data-thumbmediumwidth=\"64\" data-thumbmediumheight=\"64\" data-thumbsmallwidth=\"48\" data-thumbsmallheight=\"48\" data-imgtitle=\"title\" data-jsfolder=\"https:\/\/research.cchmc.org\/lungimage\/wp-content\/plugins\/wonderplugin-gallery\/engine\/\" style=\"display:none;\" ><a class=\"html5galleryimglink\" href=\"https:\/\/www.youtube.com\/embed\/xQsqyvtv6dI\" data-poster=\"https:\/\/img.youtube.com\/vi\/xQsqyvtv6dI\/0.jpg\"><img decoding=\"async\" class=\"lazyload html5galleryimg html5gallery-tn-image\" src=\"data:image\/gif;base64,R0lGODlhAQABAAAAACH5BAEKAAEALAAAAAABAAEAAAICTAEAOw==\" data-orig-src=\"https:\/\/img.youtube.com\/vi\/xQsqyvtv6dI\/0.jpg\" alt=\"PLISH In situ hybridization of thick human lung tissue For SFTPC, MIR29-1, and AXIN2\" data-description=\"Postnatal Day 28\"><\/a><div class=\"html5gallery-info\"><div class=\"html5gallery-title\">PLISH In situ hybridization of thick human lung tissue For SFTPC, MIR29-1, and AXIN2<\/div><div class=\"html5gallery-description\">Postnatal Day 28<\/div><\/div><\/div><\/div>\n<\/div><div class=\"fusion-clearfix\"><\/div><\/div><\/div><div class=\"fusion-layout-column fusion_builder_column fusion-builder-column-1 fusion_builder_column_1_1 1_1 fusion-one-full fusion-column-first fusion-column-last\" style=\"--awb-bg-size:cover;--awb-margin-bottom:25px;\"><div class=\"fusion-column-wrapper fusion-flex-column-wrapper-legacy\"><div class=\"fusion-text fusion-text-3\"><\/div><div class=\"fusion-tabs fusion-tabs-1 classic vertical-tabs icon-position-left mobile-mode-accordion\" style=\"--awb-title-border-radius-top-left:0px;--awb-title-border-radius-top-right:0px;--awb-title-border-radius-bottom-right:0px;--awb-title-border-radius-bottom-left:0px;--awb-inactive-color:#ebeaea;--awb-background-color:#ffffff;--awb-border-color:#ebeaea;--awb-active-border-color:#1a80b6;\"><div class=\"nav\"><ul class=\"nav-tabs\" role=\"tablist\"><li class=\"active\" role=\"presentation\"><a class=\"tab-link\" data-toggle=\"tab\" role=\"tab\" aria-controls=\"tab-065747019f497e194b8\" aria-selected=\"true\" id=\"fusion-tab-tissuepreparation\" href=\"#tab-065747019f497e194b8\"><h4 class=\"fusion-tab-heading\">Tissue Preparation<\/h4><\/a><\/li><li role=\"presentation\"><a class=\"tab-link\" data-toggle=\"tab\" role=\"tab\" aria-controls=\"tab-f39b0ab6f6eedb3ceae\" aria-selected=\"false\" tabindex=\"-1\" id=\"fusion-tab-clearing\" href=\"#tab-f39b0ab6f6eedb3ceae\"><h4 class=\"fusion-tab-heading\">Clearing<\/h4><\/a><\/li><li role=\"presentation\"><a class=\"tab-link\" data-toggle=\"tab\" role=\"tab\" aria-controls=\"tab-bd8033c67d661130bc8\" aria-selected=\"false\" tabindex=\"-1\" id=\"fusion-tab-secondharmonicimaging\" href=\"#tab-bd8033c67d661130bc8\"><h4 class=\"fusion-tab-heading\">Second Harmonic Imaging<\/h4><\/a><\/li><\/ul><\/div><div class=\"tab-content\"><div class=\"nav fusion-mobile-tab-nav\"><ul class=\"nav-tabs\" role=\"tablist\"><li class=\"active\" role=\"presentation\"><a class=\"tab-link\" data-toggle=\"tab\" role=\"tab\" aria-controls=\"tab-065747019f497e194b8\" aria-selected=\"true\" id=\"mobile-fusion-tab-tissuepreparation\" href=\"#tab-065747019f497e194b8\"><h4 class=\"fusion-tab-heading\">Tissue Preparation<\/h4><\/a><\/li><\/ul><\/div><div class=\"tab-pane fade fusion-clearfix in active\" role=\"tabpanel\" tabindex=\"0\" aria-labelledby=\"fusion-tab-tissuepreparation\" id=\"tab-065747019f497e194b8\">\n<p>Postnatal Day 28 mouse lungs were inflation fixed with 4% PFA and incubated overnight at 4<sup>o<\/sup>C. After overnight fixation, lungs were washed several times DEPC-treated 1X PBS and dehydrated through a Methanol (MeOH) series to 100% MeOH. Lung tissue was then washed in 3 changes of 100% MeOH for 30 minutes. Once tissue is equilibrated in 100% MeOH, remove air bubbles by squeezing out the air bubbles or by using a vacuum.<\/p>\n<\/div><div class=\"nav fusion-mobile-tab-nav\"><ul class=\"nav-tabs\" role=\"tablist\"><li role=\"presentation\"><a class=\"tab-link\" data-toggle=\"tab\" role=\"tab\" aria-controls=\"tab-f39b0ab6f6eedb3ceae\" aria-selected=\"false\" tabindex=\"-1\" id=\"mobile-fusion-tab-clearing\" href=\"#tab-f39b0ab6f6eedb3ceae\"><h4 class=\"fusion-tab-heading\">Clearing<\/h4><\/a><\/li><\/ul><\/div><div class=\"tab-pane fade fusion-clearfix\" role=\"tabpanel\" tabindex=\"0\" aria-labelledby=\"fusion-tab-clearing\" id=\"tab-f39b0ab6f6eedb3ceae\">\n<p>Clear in BA:BB 1:2\u00a0 (1 part benzyl alcohol: 2 parts benzyl benzoate). For a 1.5cm x 1.5 cm x 5mm piece of tissue, it takes ~ 1 hour to clear.<\/p>\n<\/div><div class=\"nav fusion-mobile-tab-nav\"><ul class=\"nav-tabs\" role=\"tablist\"><li role=\"presentation\"><a class=\"tab-link\" data-toggle=\"tab\" role=\"tab\" aria-controls=\"tab-bd8033c67d661130bc8\" aria-selected=\"false\" tabindex=\"-1\" id=\"mobile-fusion-tab-secondharmonicimaging\" href=\"#tab-bd8033c67d661130bc8\"><h4 class=\"fusion-tab-heading\">Second Harmonic Imaging<\/h4><\/a><\/li><\/ul><\/div><div class=\"tab-pane fade fusion-clearfix\" role=\"tabpanel\" tabindex=\"0\" aria-labelledby=\"fusion-tab-secondharmonicimaging\" id=\"tab-bd8033c67d661130bc8\">\n<p>Transfer lungs to fresh BA:BB. Image on a multiphoton microscope with glycerol immersion objectives. Mount tissue in BA:BB in 3D printed sample holders and use a coverglass bottom dish as a lid for the sample. The coverglass bottom dish is filled with glycerol to match the refractive index of the sample. We use either a 10X NA 0.5 WD 5.5mm or a 20X NA 1.0 WD 8.2mm.\u00a0 Image using backscattered SHG with GaAsP NDDs using 840nm excitation and 420\/20 filter for SHG, 525\/50 for autofluorescence and a 585\/50 for pigment (pigment, carbon particles and other debris will \u201cexplode\u201d under IR excitation and needs to be captured to remove from final datasets). Mount tissue in BA:BB in 3D printed sample holders and use a coverglass bottom dish as a lid for the sample. The coverglass bottom dish is filled with glycerol to match the refractive index of the sample.<\/p>\n<\/div><\/div><\/div><div class=\"fusion-clearfix\"><\/div><\/div><\/div><\/div><\/div>\n","protected":false},"excerpt":{"rendered":"","protected":false},"author":1,"featured_media":0,"comment_status":"closed","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"footnotes":""},"categories":[274,80,1],"tags":[],"class_list":["post-19433","post","type-post","status-publish","format-standard","hentry","category-c57bl6","category-p28","category-uncategorized"],"yoast_head":"<!-- This site is optimized with the Yoast SEO plugin v25.6 - https:\/\/yoast.com\/wordpress\/plugins\/seo\/ -->\n<title>Postnatal Day 28 Mouse Lung Second Harmonic Imaging -<\/title>\n<meta name=\"robots\" content=\"index, follow, max-snippet:-1, max-image-preview:large, max-video-preview:-1\" \/>\n<link rel=\"canonical\" href=\"https:\/\/research.cchmc.org\/lungimage\/?p=19433\" \/>\n<meta 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