{"id":6552,"date":"2016-04-04T18:50:01","date_gmt":"2016-04-04T18:50:01","guid":{"rendered":"https:\/\/research.cchmc.org\/lungimage\/?p=6552"},"modified":"2018-08-19T23:52:54","modified_gmt":"2018-08-19T23:52:54","slug":"pnd14-c57bl6-sftpc-nkx2-1-and-acta2-confocal-imaging","status":"publish","type":"post","link":"https:\/\/research.cchmc.org\/lungimage\/?p=6552","title":{"rendered":"Postnatal Day 14 C57BL6 SFTPC, NKX2.1, and ACTA2 Confocal Imaging"},"content":{"rendered":"

\"Post<\/a>
Post Natal Day 14 Confocal Imaging Showing Protein Expression of Sftpc, Nkx2.1, and Acta2 Genes<\/div>
20x Tile Scan<\/div><\/div>\"Post<\/a>
Post Natal Day 14 Confocal Imaging Showing Protein Expression of Sftpc, Nkx2.1, and Acta2 Genes<\/div>
20x<\/div><\/div>\"Post<\/a>
Post Natal Day 14 Confocal Imaging Showing Protein Expression of Sftpc, Nkx2.1, and Acta2 Genes<\/div>
60x<\/div><\/div>\"Post<\/a>
Post Natal Day 14 Confocal Imaging Showing Protein Expression of Sftpc, Nkx2.1, and Acta2 Genes<\/div>
20x<\/div><\/div>\"Post<\/a>
Post Natal Day 14 Confocal Imaging Showing Protein Expression of Sftpc, Nkx2.1, and Acta2 Genes<\/div>
60x<\/div><\/div>\"Post<\/a>
Post Natal Day 14 Confocal Imaging Showing Protein Expression of Sftpc, Nkx2.1, and Acta2 Genes<\/div>
60x<\/div><\/div>\"Post<\/a>
Post Natal Day 14 Confocal Imaging Showing Protein Expression of Sftpc, Nkx2.1, and Acta2 Genes<\/div>
20x<\/div><\/div>\"Post<\/a>
Post Natal Day 14 Confocal Imaging Showing Protein Expression of Sftpc, Nkx2.1, and Acta2 Genes<\/div>
60x<\/div><\/div>\"Post<\/a>
Post Natal Day 14 Confocal Imaging Showing Protein Expression of Sftpc, Nkx2.1, and Acta2 Genes<\/div>
20x Tile Scan<\/div><\/div>\"Post<\/a>
Post Natal Day 14 Confocal Imaging Showing Protein Expression of Sftpc, Nkx2.1, and Acta2 Genes<\/div>
20x<\/div><\/div>\"Post<\/a>
Post Natal Day 14 Confocal Imaging Showing Protein Expression of Sftpc, Nkx2.1, and Acta2 Genes<\/div>
60x<\/div><\/div>\"Post<\/a>
Post Natal Day 14 Confocal Imaging Showing Protein Expression of Sftpc, Nkx2.1, and Acta2 Genes<\/div>
60x<\/div><\/div>\"Post<\/a>
Post Natal Day 14 Confocal Imaging Showing Protein Expression of Sftpc, Nkx2.1, and Acta2 Genes<\/div>
20x<\/div><\/div>\"Post<\/a>
Post Natal Day 14 Confocal Imaging Showing Protein Expression of Sftpc, Nkx2.1, and Acta2 Genes<\/div>
60x<\/div><\/div>\"Post<\/a>
Post Natal Day 14 Confocal Imaging Showing Protein Expression of Sftpc, Nkx2.1, and Acta2 Genes<\/div>
60x<\/div><\/div><\/div><\/div>\n<\/div>
<\/div><\/div><\/div><\/div><\/div>
<\/i>Single Cell RNA-Seq and Gene Info <\/span><\/a><\/div>
<\/div><\/div><\/div>\n
<\/div><\/div><\/div>
<\/div><\/div><\/div><\/div><\/div>\n

Immunofluorescence- SFTPC<\/strong>,<\/strong> NKX2.1<\/strong>, and ACTA2<\/strong><\/a><\/span><\/p>\n

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

Purpose: \u00a0<\/strong>Immunofluorescence on slides of 7\u00b5m frozen sections of C57Bl\/6 PND14 lungs for NKX2.1, <\/strong>SFTPC, <\/strong>and ACTA2<\/strong><\/a> with antigen retrieval.<\/p>\n

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

    \n
  1. Paraffin sections are placed at 50o<\/sup>C for 2 hours to overnight to melt paraffin. 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.<\/li>\n<\/ol>\n
      \n
    1. Briefly equilibrate slides in Antigen Retrieval Buffer.<\/li>\n
    2. Antigen retrieval, pH 6.0 (times will vary according to microwave).<\/li>\n
    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>\n
    4. Microwave according to instructions on microwave.<\/li>\n
    5. Cool on countertop, 15 min.<\/li>\n
    6. Rinse with dH2<\/sub>O<\/li>\n
    7. 1X PBS, 5 min.<\/li>\n
    8. Block in 4% Donkey serum\/PBS-T, 2 hours at RT.<\/li>\n
    9. For Rabbit anti-SFTPC<\/strong> (RB458, BL 8\/24\/11, in house) dilute 1:1000, 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>\n<\/ol>\n

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

        \n
      1. Rinse slides in PBS-T 3X, 5 min.<\/li>\n
      2. Apply secondary antibody, Goat Alexa Fluor 488 anti-rabbit IgG (A11034<\/strong><\/a>, Lot# 1298480,\u00a0 for anti-SFTPC<\/strong>), Goat Alexa Fluor 555 anti-guinea pig IgG (A21435<\/strong><\/a>, Lot# 1571699, for 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>\n
      3. Rinse in PBS-T 3X, 5 min.<\/li>\n
      4. Dilute DAPI 1:2000 and apply to slides for 10 min.<\/li>\n
      5. Wash in PBS-T 3X, 5 min.<\/li>\n
      6. Rinse slides in 0.1M PB 3X for 5 min.<\/li>\n
      7. Add 1 drop of Prolong Gold anti-fade mounting medium (P36930).<\/li>\n
      8. Coverslip with Gold Seal Coverslip (Cat# 3422 Electron Microscopy Sciences, 22 X 22 mm).<\/li>\n
      9. Allow Prolong Gold to cure overnight at room temp in light sealed box.<\/li>\n<\/ol>\n

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

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

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

        Tissue Used:<\/strong> \u00a0\u00a0 <\/strong>LMM.15.34A.5L.38<\/strong><\/p>\n

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

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

        Weight: 5.91g<\/strong><\/p>\n<\/div>

        <\/div><\/div><\/div><\/div><\/div><\/p>\n","protected":false},"excerpt":{"rendered":"","protected":false},"author":1,"featured_media":6553,"comment_status":"closed","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"footnotes":""},"categories":[274,107],"tags":[5,6,22,51,29,121,36],"class_list":["post-6552","post","type-post","status-publish","format-standard","has-post-thumbnail","hentry","category-c57bl6","category-p14-confocal","tag-acta2","tag-alpha-smooth-muscle-actin","tag-nkx2-1","tag-pro-spc","tag-sftpc","tag-sma","tag-ttf-1"],"yoast_head":"\nPostnatal Day 14 C57BL6 SFTPC, 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=6552\" \/>\n<meta property=\"og:locale\" content=\"en_US\" \/>\n<meta property=\"og:type\" content=\"article\" \/>\n<meta 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