{"id":1997,"date":"2015-04-03T00:12:05","date_gmt":"2015-04-03T00:12:05","guid":{"rendered":"https:\/\/research.cchmc.org\/lungimage\/?p=1997"},"modified":"2018-08-17T22:42:22","modified_gmt":"2018-08-17T22:42:22","slug":"e18-5-c57bl6-sox9-nkx2-1-and-acta2-confocal-imaging","status":"publish","type":"post","link":"https:\/\/research.cchmc.org\/lungimage\/?p=1997","title":{"rendered":"E18.5 C57Bl6 SOX9, NKX2.1, and ACTA2 Confocal Imaging"},"content":{"rendered":"\t\t
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E18.5 C57Bl6 SOX9, 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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\"E18.5<\/a>
E18.5 Confocal Imaging Showing Protein Expression of Sox9, Nkx2.1, and Acta2 Genes<\/div>
20X Tile Scan<\/div><\/div>\"E18.5<\/a>
E18.5 Confocal Imaging Showing Protein Expression of Sox9, Nkx2.1, and Acta2 Genes<\/div>
20X<\/div><\/div>\"E18.5<\/a>
E18.5 Confocal Imaging Showing Protein Expression of Sox9, Nkx2.1, and Acta2 Genes<\/div>
60X<\/div><\/div>\"E18.5<\/a>
E18.5 Confocal Imaging Showing Protein Expression of Sox9, Nkx2.1, and Acta2 Genes<\/div>
20X<\/div><\/div>\"E18.5<\/a>
E18.5 Confocal Imaging Showing Protein Expression of Sox9, Nkx2.1, and Acta2 Genes<\/div>
60X<\/div><\/div>\"E18.5<\/a>
E18.5 Confocal Imaging Showing Protein Expression of Sox9, Nkx2.1, and Acta2 Genes<\/div>
20X<\/div><\/div>\"E18.5<\/a>
E18.5 Confocal Imaging Showing Protein Expression of Sox9, Nkx2.1, and Acta2 Genes<\/div>
60X<\/div><\/div>\"E18.5<\/a>
E18.5 Confocal Imaging Showing Protein Expression of Sox9, Nkx2.1, and Acta2 Genes<\/div>
20X<\/div><\/div>\"E18.5<\/a>
E18.5 Confocal Imaging Showing Protein Expression of Sox9, Nkx2.1, and Acta2 Genes<\/div>
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Immunofluorescence- <\/strong>SOX9<\/strong><\/a>, NKX2.1 <\/strong>and ACTA2<\/strong><\/a><\/span><\/p>

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

Purpose:\u00a0 <\/strong>Immunofluorescence on slides of 7\u00b5m frozen sections of C57BL6 E18.5lungs for SOX9<\/strong><\/a>, NKX2.1 <\/strong>and ACTA2<\/strong><\/a> with antigen retrieval.<\/p>

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

  1. For Frozen tissue, rinse 2X in PBS, then 5 min in 4% PFA\/PBS, then rinse 1X in PBS.<\/li>
  2. Briefly equilibrate slides in Antigen Retrieval Buffer.<\/li>
  3. Antigen retrieval, pH 6.0 (times will vary according to microwave).<\/li>
  4. 10 mM sodium citrate, pH 6.0, and heat in a microwave at 96o<\/sup>C. *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>
  5. Microwave according to instructions on microwave.<\/li>
  6. Cool on countertop, 15 min.<\/li>
  7. Rinse with dH2<\/sub>O<\/li>
  8. 1X PBS, 5 min.<\/li>
  9. Block in 4% Goat serum\/PBS-T, 2 hours at RT.<\/li>
  10. For Rabbit anti-SOX9<\/strong><\/a> (AB-5535, Lot # 2167153, Millipore) dilute 1:100, For guinea pig anti-NKX2.1 (G237, Bl. 10\/7\/11) dilute 1:500, for mouse anti- ACTA2<\/strong><\/a> (A5228, \u03b1 Smooth muscle actin<\/strong><\/a>, Sigma) dilute 1:2000 in blocking buffer. Spin down antibody cocktail in a \u00b5fuge for 10 min and incubate on tissue overnight @ 4o<\/sup>C.
    <\/span><\/li><\/ol>

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

    1. Rinse slides in PBS-T 3X, 5 min.<\/li>
    2. Apply secondary antibody, Goat Alexa Fluor 488 anti-rabbit IgG (A11034<\/strong><\/a>, Lot# 1298480, for SOX9<\/strong><\/a>), Goat Alexa Fluor 555 anti-guinea pig IgG (A21435<\/strong><\/a>, Lot# 1571699, GPTTF-1) at 1:200 at 1:200, Donkey Alexa Fluor 647 anti-mouse IgG (A21136<\/strong><\/a>, Lot# 1345055, anti-ACTA2<\/strong><\/a>) at 1:200, in blocking buffer. Spin down antibody cocktail in a \u00b5fuge for 10 min 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 or TB, 3X, 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 slides @ 4o<\/sup>C.<\/li><\/ol>

      Tissue Used:<\/strong><\/p>

      LMM.14.38.2.32 E18.5<\/p>

      Gender: Male<\/p>

      Crown-to-Rump Length: 21.0 mm<\/p>

      Weight=1.06g<\/p>

      Appendix<\/strong><\/p>

      Antigen Retrieval Solution<\/strong>
      18ml Solution A- 0.1M Citric Acid (Sigma, C1909) pH 2.5
      82ml Reagent B- 0.1M Sodium Citrate (Sigma, S4641 ) pH 8.2
      1L dH2O
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      Immunofluorescence- SOX9, NKX2.1 and ACTA2 \u00a0 Purpose:\u00a0 Immunofluorescence on slides of 7\u00b5m frozen sections of C57BL6 E18.5lungs for SOX9, NKX2.1 and ACTA2 with antigen retrieval. Day 1 For Frozen tissue, rinse 2X in PBS, then 5 min in 4% PFA\/PBS, then rinse 1X in PBS. Briefly equilibrate slides in Antigen Retrieval Buffer. Antigen retrieval, pH […]<\/p>\n","protected":false},"author":1,"featured_media":26026,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"footnotes":""},"categories":[83,274],"tags":[5,6,22,32],"class_list":["post-1997","post","type-post","status-publish","format-standard","has-post-thumbnail","hentry","category-e18-5","category-c57bl6","tag-acta2","tag-alpha-smooth-muscle-actin","tag-nkx2-1","tag-sox9"],"yoast_head":"\nE18.5 C57Bl6 SOX9, 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=1997\" \/>\n<meta property=\"og:locale\" content=\"en_US\" \/>\n<meta property=\"og:type\" content=\"article\" \/>\n<meta property=\"og:title\" content=\"E18.5 C57Bl6 SOX9, NKX2.1, and ACTA2 Confocal Imaging -\" \/>\n<meta property=\"og:description\" content=\"Immunofluorescence- SOX9, NKX2.1 and ACTA2 \u00a0 Purpose:\u00a0 Immunofluorescence on slides of 7\u00b5m frozen sections of C57BL6 E18.5lungs for SOX9, NKX2.1 and ACTA2 with antigen retrieval. Day 1 For Frozen tissue, rinse 2X in PBS, then 5 min in 4% PFA\/PBS, then rinse 1X in PBS. Briefly equilibrate slides in Antigen Retrieval Buffer. 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