{"id":6597,"date":"2016-04-06T17:40:38","date_gmt":"2016-04-06T17:40:38","guid":{"rendered":"https:\/\/research.cchmc.org\/lungimage\/?p=6597"},"modified":"2018-08-14T19:23:37","modified_gmt":"2018-08-14T19:23:37","slug":"pnd14-c57bl6-sox9-sftpc-and-acta2-confocal-imaging","status":"publish","type":"post","link":"https:\/\/research.cchmc.org\/lungimage\/?p=6597","title":{"rendered":"Postnatal Day 14 C57BL6 SOX9, SFTPC, and ACTA2 Confocal Imaging"},"content":{"rendered":"\t\t
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Postnatal Day 14 C57BL6 SOX9, SFTPC, 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 Sox9, Sftpc, and Acta2 Genes<\/div>
20x<\/div><\/div>\"Post<\/a>
Post Natal Day 14 Confocal Imaging Showing Protein Expression of Sox9, Sftpc, and Acta2 Genes<\/div>
60x<\/div><\/div>\"Post<\/a>
Post Natal Day 14 Confocal Imaging Showing Protein Expression of Sox9, Sftpc, and Acta2 Genes<\/div>
20x<\/div><\/div>\"Post<\/a>
Post Natal Day 14 Confocal Imaging Showing Protein Expression of Sox9, Sftpc, and Acta2 Genes<\/div>
60x<\/div><\/div>\"Post<\/a>
Post Natal Day 14 Confocal Imaging Showing Protein Expression of Sox9, Sftpc, and Acta2 Genes<\/div>
20x<\/div><\/div>\"Post<\/a>
Post Natal Day 14 Confocal Imaging Showing Protein Expression of Sox9, Sftpc, and Acta2 Genes<\/div>
60x<\/div><\/div>\"Post<\/a>
Post Natal Day 14 Confocal Imaging Showing Protein Expression of Sox9, Sftpc, and Acta2 Genes<\/div>
20x<\/div><\/div>\"Post<\/a>
Post Natal Day 14 Confocal Imaging Showing Protein Expression of Sox9, Sftpc, and Acta2 Genes<\/div>
60x<\/div><\/div>\"Post<\/a>
Post Natal Day 14 Confocal Imaging Showing Protein Expression of Sox9, Sftpc, and Acta2 Genes<\/div>
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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.\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>
  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% Donkey serum\/PBS-T, 2 hours at RT.<\/li>
  10. For Rabbit anti-SOX9<\/strong><\/a> (AB<\/strong>–5535<\/strong><\/a>, Lot # 2167153, Millipore) dilute 1:100, For goat anti-SFTPC<\/strong><\/a> (SC-7706, Santa Cruz) dilute 1:100, 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<\/strong><\/a>, Lot# 1608521, for anti-SOX9<\/strong><\/a>) at 1:200, Donkey Alexa Fluor 568 anti-goat IgG (A11057<\/strong><\/a>, Lot# 1485187, for anti-SFTPC<\/strong><\/a>) at 1:200, Donkey Alexa Fluor 647 anti-mouse IgG A31571<\/strong><\/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><\/ol>
      1. Rinse in PBS-T 3X, 5 min.<\/li>
      2. Dilute DAPI 1:2000 and apply to slides for 15 min.<\/li>
      3. Wash in PBS-T 3X, 5 min.<\/li>
      4. Rinse slides in 0.1M PB or TB, 3X, 5 min.<\/li>
      5. Add 1 drop of Prolong Gold anti-fade mounting medium (P36930).<\/li>
      6. Coverslip with Gold Seal Coverslip (Cat# 3422 Electron Microscopy Sciences, 22 X 22 mm).<\/li>
      7. Allow Prolong Gold to cure overnight at room temp in light sealed box.<\/li>
      8. Store slides in light sealed box @ 4o<\/sup>C<\/li><\/ol>

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

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        Tissue Used: \u00a0\u00a0 <\/strong>LMM.15.34A.5L.48<\/strong><\/p>

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

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

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

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

        Appendix<\/span><\/p>

        \u00a0<\/span>Antigen Retrieval Solution<\/span><\/p>

        18ml Solution A- 0.1M Citric Acid (Sigma, C1909) pH 2.5<\/p>

        82ml Reagent B- 0.1M Sodium Citrate (Sigma, S4641 ) pH 8.2<\/p>

        1L dH2<\/sub>O<\/p>

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