{"id":6556,"date":"2016-04-04T18:57:12","date_gmt":"2016-04-04T18:57:12","guid":{"rendered":"https:\/\/research.cchmc.org\/lungimage\/?p=6556"},"modified":"2018-08-19T23:23:09","modified_gmt":"2018-08-19T23:23:09","slug":"pnd14-c57bl6-hopx-nkx2-1-and-acta2-confocal-imaging","status":"publish","type":"post","link":"https:\/\/research.cchmc.org\/lungimage\/?p=6556","title":{"rendered":"Postnatal Day 14 C57BL6 HOPX, NKX2.1, and ACTA2 Confocal Imaging"},"content":{"rendered":"\t\t
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Postnatal Day 14 C57BL6 HOPX, NKX2.1, and ACTA2 Confocal Imaging<\/h1>\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t
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\"Post<\/a>
Post Natal Day 14 Confocal Imaging Showing Protein Expression of Hopx, Nkx2.1, and Acta2 Genes<\/div>
20x Tile Scan<\/div><\/div>\"Post<\/a>
Post Natal Day 14 Confocal Imaging Showing Protein Expression of Hopx, Nkx2.1, and Acta2 Genes<\/div>
20x<\/div><\/div>\"Post<\/a>
Post Natal Day 14 Confocal Imaging Showing Protein Expression of Hopx, Nkx2.1, and Acta2 Genes<\/div>
60x<\/div><\/div>\"Post<\/a>
Post Natal Day 14 Confocal Imaging Showing Protein Expression of Hopx, Nkx2.1, and Acta2 Genes<\/div>
20x Tile Scan<\/div><\/div>\"Post<\/a>
Post Natal Day 14 Confocal Imaging Showing Protein Expression of Hopx, Nkx2.1, and Acta2 Genes<\/div>
20x<\/div><\/div>\"Post<\/a>
Post Natal Day 14 Confocal Imaging Showing Protein Expression of Hopx, Nkx2.1, and Acta2 Genes<\/div>
60x<\/div><\/div>\"Post<\/a>
Post Natal Day 14 Confocal Imaging Showing Protein Expression of Hopx, Nkx2.1, and Acta2 Genes<\/div>
20x<\/div><\/div>\"Post<\/a>
Post Natal Day 14 Confocal Imaging Showing Protein Expression of Hopx, Nkx2.1, and Acta2 Genes<\/div>
60x<\/div><\/div>\"Post<\/a>
Post Natal Day 14 Confocal Imaging Showing Protein Expression of Hopx, Nkx2.1, and Acta2 Genes<\/div>
60x<\/div><\/div>\"Post<\/a>
Post Natal Day 14 Confocal Imaging Showing Protein Expression of Hopx, Nkx2.1, and Acta2 Genes<\/div>
20x<\/div><\/div>\"Post<\/a>
Post Natal Day 14 Confocal Imaging Showing Protein Expression of Hopx, Nkx2.1, and Acta2 Genes<\/div>
60x<\/div><\/div>\"Post<\/a>
Post Natal Day 14 Confocal Imaging Showing Protein Expression of Hopx, Nkx2.1, and Acta2 Genes<\/div>
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Immunofluorescence- <\/strong>HOPX<\/strong><\/a>, NKX2.1, and <\/strong>ACTA2<\/strong><\/a><\/p>

\u00a0<\/strong><\/p>

Purpose:<\/strong>\u00a0 <\/strong>Stain slides of frozen sections of C57Bl\/6 PND14 lung for HOPX<\/strong><\/a>, NKX2.1, and <\/strong>ACTA2<\/strong><\/a> with antigen retrieval.<\/p>

Day 1<\/strong><\/p>

  1. For Frozen tissue remove OCT and rehydrate by washing in 2X in PBS, then 5 min in 4% PFA\/PBS, then rinse 1X in PBS. Briefly equilibrate slides in Antigen Retrieval Buffer.<\/li>
  2. Antigen retrieval, pH 6.0 (times will vary according to microwave).<\/li>
  3. 10 mM sodium citrate, pH 6.0, and heat in a microwave at 96o<\/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 dH2<\/sub>O).<\/li>
  4. Microwave according to instructions on microwave.<\/li>
  5. Cool on countertop, 15 min.<\/li>
  6. Rinse with dH2<\/sub>O<\/li>
  7. 1X PBS, 5 min.<\/li>
  8. Block in 4% Donkey serum\/PBS-T, 2 hours at RT.<\/li>
  9. For Rabbit anti-HOPX<\/strong><\/a> (SC-30216<\/a>, Lot # C2212, Santa Cruz) dilute 1:200, For guinea pig anti-NKX2.1 (G237, Bl. 10\/7\/11) dilute 1:200, for mouse anti-ACTA2<\/strong><\/a> (\u03b1 Smooth muscle actin<\/strong><\/a>, A5228, Sigma)\u00a0dilute 1:2000 in blocking buffer. Spin down in \u00b5fuge for 10 minutes and incubate on tissue overnight @ 4o<\/sup>C.<\/li><\/ol>

    Day 2 <\/strong><\/p>

    1. Rinse slides in PBS-T 3X, 5 min.<\/li>
    2. Apply secondary antibody , Donkey Alexa Fluor 488 anti-rabbit IgG (A21206<\/a>, Lot# 1608521, for anti-HOPX<\/strong><\/a>), Goat Alexa Fluor 555 anti-guinea pig IgG (A21435<\/strong><\/a>, Lot# 1571699, anti-NKX2.1<\/strong>) at 1:200, Donkey Alexa Fluor 647 anti-mouse IgG A31571<\/a>, Lot# 1549801, ACTA2<\/strong><\/a>) at 1:200, in blocking buffer. Spin down in \u00b5fuge for 10 min, apply to tissue and incubated at room temperature for 1 hour.<\/li>
    3. Rinse in PBS-T 3X, 5 min.<\/li>
    4. Dilute DAPI 1:2000 and apply to slides for 10 min.<\/li>
    5. Wash in PBS-T 3X, 5 min.<\/li>
    6. Rinse slides in 0.1M PB 3X for 5 min.<\/li>
    7. Add 1 drop of Prolong Gold anti-fade mounting medium (P36930).<\/li>
    8. Coverslip with Gold Seal Coverslip (Cat# 3422 Electron Microscopy Sciences, 22 X 22 mm).<\/li>
    9. Allow Prolong Gold to cure overnight at room temp in light sealed box.<\/li>
    10. Store in a light sealed box @ 4o<\/sup>C.<\/li><\/ol>

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      Tissue Used: \u00a0\u00a0 <\/strong>LMM.15.34A.5L.52<\/strong><\/p>

      C57BL6 PND14 <\/strong><\/p>

      Gender: Male <\/strong><\/p>

      Weight: 5.91g<\/strong><\/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

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      Immunofluorescence- HOPX, NKX2.1, and ACTA2 \u00a0 Purpose:\u00a0 Stain slides of frozen sections of C57Bl\/6 PND14 lung for HOPX, NKX2.1, and ACTA2 with antigen retrieval. Day 1 For Frozen tissue remove OCT and rehydrate by washing in 2X in PBS, then 5 min in 4% PFA\/PBS, then rinse 1X in PBS. Briefly equilibrate slides in Antigen […]<\/p>\n","protected":false},"author":1,"featured_media":6555,"comment_status":"closed","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"footnotes":""},"categories":[274,107],"tags":[5,6,19,22,121,36],"class_list":["post-6556","post","type-post","status-publish","format-standard","has-post-thumbnail","hentry","category-c57bl6","category-p14-confocal","tag-acta2","tag-alpha-smooth-muscle-actin","tag-hopx","tag-nkx2-1","tag-sma","tag-ttf-1"],"yoast_head":"\nPostnatal Day 14 C57BL6 HOPX, NKX2.1, and ACTA2 Confocal 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=6556\" \/>\n<meta property=\"og:locale\" content=\"en_US\" \/>\n<meta property=\"og:type\" content=\"article\" \/>\n<meta property=\"og:title\" content=\"Postnatal Day 14 C57BL6 HOPX, NKX2.1, and ACTA2 Confocal Imaging -\" \/>\n<meta property=\"og:description\" content=\"Immunofluorescence- HOPX, NKX2.1, and ACTA2 \u00a0 Purpose:\u00a0 Stain slides of frozen sections of C57Bl\/6 PND14 lung for HOPX, NKX2.1, and ACTA2 with antigen retrieval. Day 1 For Frozen tissue remove OCT and rehydrate by washing in 2X in PBS, then 5 min in 4% PFA\/PBS, then rinse 1X in PBS. Briefly equilibrate slides in Antigen […]\" \/>\n<meta property=\"og:url\" content=\"https:\/\/research.cchmc.org\/lungimage\/?p=6556\" \/>\n<meta property=\"article:published_time\" content=\"2016-04-04T18:57:12+00:00\" \/>\n<meta property=\"article:modified_time\" content=\"2018-08-19T23:23:09+00:00\" \/>\n<meta property=\"og:image\" content=\"https:\/\/research.cchmc.org\/lungimage\/wp-content\/uploads\/2016\/04\/2015-10-19_C57BL6_PND14_LMM-15-34A.5L-43_HOPX_NKX2.1_ACTA2-12-1.jpg\" \/>\n\t<meta property=\"og:image:width\" content=\"300\" \/>\n\t<meta property=\"og:image:height\" content=\"300\" \/>\n\t<meta property=\"og:image:type\" content=\"image\/jpeg\" \/>\n<meta name=\"author\" content=\"kitc5i\" \/>\n<meta name=\"twitter:card\" content=\"summary_large_image\" \/>\n<meta name=\"twitter:label1\" content=\"Written by\" \/>\n\t<meta name=\"twitter:data1\" content=\"kitc5i\" \/>\n\t<meta name=\"twitter:label2\" content=\"Est. reading time\" \/>\n\t<meta name=\"twitter:data2\" 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