{"id":5922,"date":"2016-03-14T18:32:27","date_gmt":"2016-03-14T18:32:27","guid":{"rendered":"https:\/\/research.cchmc.org\/lungimage\/?p=5922"},"modified":"2018-08-17T23:27:08","modified_gmt":"2018-08-17T23:27:08","slug":"pnd01-c57bl6-hopx-nkx2-1-and-acta2-confocal-imaging","status":"publish","type":"post","link":"https:\/\/research.cchmc.org\/lungimage\/?p=5922","title":{"rendered":"Postnatal Day 1 C57BL6 HOPX, NKX2.1, and ACTA2 Confocal Imaging"},"content":{"rendered":"\t\t
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Postnatal Day 1 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 1 Confocal Imaging Showing Protein Expression of Hopx, Nkx2.1, and Acta2 Genes<\/div>
20x Tile Scan<\/div><\/div>\"Post<\/a>
Post Natal Day 1 Confocal Imaging Showing Protein Expression of Hopx, Nkx2.1, and Acta2 Genes<\/div>
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Post Natal Day 1 Confocal Imaging Showing Protein Expression of Hopx, Nkx2.1, and Acta2 Genes<\/div>
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Post Natal Day 1 Confocal Imaging Showing Protein Expression of Hopx, Nkx2.1, and Acta2 Genes<\/div>
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Post Natal Day 1 Confocal Imaging Showing Protein Expression of Hopx, Nkx2.1, and Acta2 Genes<\/div>
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Post Natal Day 1 Confocal Imaging Showing Protein Expression of Hopx, Nkx2.1, and Acta2 Genes<\/div>
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Post Natal Day 1 Confocal Imaging Showing Protein Expression of Hopx, Nkx2.1, and Acta2 Genes<\/div>
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Post Natal Day 1 Confocal Imaging Showing Protein Expression of Hopx, Nkx2.1, and Acta2 Genes<\/div>
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Post Natal Day 1 Confocal Imaging Showing Protein Expression of Hopx, Nkx2.1, and Acta2 Genes<\/div>
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Post Natal Day 1 Confocal Imaging Showing Protein Expression of Hopx, Nkx2.1, and Acta2 Genes<\/div>
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Post Natal Day 1 Confocal Imaging Showing Protein Expression of Hopx, Nkx2.1, and Acta2 Genes<\/div>
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Post Natal Day 1 Confocal Imaging Showing Protein Expression of Hopx, Nkx2.1, and Acta2 Genes<\/div>
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Post Natal Day 1 Confocal Imaging Showing Protein Expression of Hopx, Nkx2.1, and Acta2 Genes<\/div>
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Post Natal Day 1 Confocal Imaging Showing Protein Expression of Hopx, Nkx2.1, and Acta2 Genes<\/div>
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Post Natal Day 1 Confocal Imaging Showing Protein Expression of Hopx, Nkx2.1, and Acta2 Genes<\/div>
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Post Natal Day 1 Confocal Imaging Showing Protein Expression of Hopx, Nkx2.1, and Acta2 Genes<\/div>
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Post Natal Day 1 Confocal Imaging Showing Protein Expression of Hopx, Nkx2.1, and Acta2 Genes<\/div>
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Post Natal Day 1 Confocal Imaging Showing Protein Expression of Hopx, Nkx2.1, and Acta2 Genes<\/div>
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Post Natal Day 1 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 C57BL6 PND01 lung for HOPX<\/strong><\/a>, NKX2.1, and <\/strong>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.\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% Goat serum\/PBS-T, 2 hours at RT.<\/li>
  10. For rabbit anti- HOPX<\/strong><\/a> (SC-30216<\/strong><\/a>, Lot # C2212, Santa Cruz) dilute 1:200, For guinea pig anti- anti-NKX2.1<\/strong> (G237, Bl. 8\/25\/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, Goat Alexa Fluor 488 anti-rabbit IgG (A11034<\/strong><\/a>, Lot# 1298480,\u00a0 for anti- HOPX<\/strong><\/a>), 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<\/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. Spin down in \u00b5fuge for 10 min, apply to tissue and incubated at room temperature for 1 hour.<\/li>
      2. Rinse in PBS-T 3X, 5 min.<\/li>
      3. Dilute DAPI 1:2000 and apply to slides for 15 min.<\/li>
      4. Wash in PBS-T 3X, 5 min.<\/li>
      5. Rinse slides in 0.1M PB or TB, 3X, 5 min.<\/li>
      6. Add 1 drop of Prolong Gold anti-fade mounting medium (P36930).<\/li>
      7. Coverslip with Gold Seal Coverslip (Cat# 3422 Electron Microscopy Sciences, 22 X 22 mm).<\/li>
      8. Allow Prolong Gold to cure overnight at room temp in light sealed box.<\/li>
      9. Store slides in light sealed box at 4o<\/sup>C.<\/li><\/ol>

        Tissue Used:<\/strong>M.15.69A.5.34 <\/strong><\/p>

        PND01 C57BL6 <\/strong><\/p>

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

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