{"id":6643,"date":"2016-04-06T18:17:33","date_gmt":"2016-04-06T18:17:33","guid":{"rendered":"https:\/\/research.cchmc.org\/lungimage\/?p=6643"},"modified":"2018-08-14T19:23:36","modified_gmt":"2018-08-14T19:23:36","slug":"pnd14-c57bl6-hopx-sftpc-and-acta2-confocal-imaging","status":"publish","type":"post","link":"https:\/\/research.cchmc.org\/lungimage\/?p=6643","title":{"rendered":"Postnatal Day 14 C57BL6 HOPX, SFTPC and ACTA2 Confocal Imaging"},"content":{"rendered":"\t\t
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Postnatal Day 14 C57BL6 HOPX, 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 Hopx, Sftpc, and Acta2 Genes<\/div>
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Post Natal Day 14 Confocal Imaging Showing Protein Expression of Hopx, Sftpc, and Acta2 Genes<\/div>
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Post Natal Day 14 Confocal Imaging Showing Protein Expression of Hopx, Sftpc, and Acta2 Genes<\/div>
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Post Natal Day 14 Confocal Imaging Showing Protein Expression of Hopx, Sftpc, and Acta2 Genes<\/div>
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Post Natal Day 14 Confocal Imaging Showing Protein Expression of Hopx, Sftpc, and Acta2 Genes<\/div>
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Post Natal Day 14 Confocal Imaging Showing Protein Expression of Hopx, Sftpc, and Acta2 Genes<\/div>
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Post Natal Day 14 Confocal Imaging Showing Protein Expression of Hopx, Sftpc, and Acta2 Genes<\/div>
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Post Natal Day 14 Confocal Imaging Showing Protein Expression of Hopx, Sftpc, and Acta2 Genes<\/div>
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Post Natal Day 14 Confocal Imaging Showing Protein Expression of Hopx, Sftpc, and Acta2 Genes<\/div>
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Post Natal Day 14 Confocal Imaging Showing Protein Expression of Hopx, Sftpc, and Acta2 Genes<\/div>
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Post Natal Day 14 Confocal Imaging Showing Protein Expression of Hopx, Sftpc, and Acta2 Genes<\/div>
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Immunofluorescence- <\/strong>HOPX<\/strong><\/a>, <\/strong>SFTPC<\/strong><\/a>, <\/strong>and ACTA2<\/strong><\/a><\/span><\/p>

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

Purpose:<\/strong> Stain slides of 5\u00b5m paraffin sections of C57BL\/6J PND14 lungs for HOPX<\/strong><\/a>,<\/strong> SFTPC<\/strong><\/a>, <\/strong>and ACTA2<\/strong><\/a> with antigen retrieval.<\/p>

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

  1. Paraffin sections are placed at 60o<\/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>
  2. Briefly equilibrate slides in Antigen Retrieval Buffer.<\/li><\/ol>
    1. 10 mM sodium citrate, pH 6.0, and heat in a microwave for 15 min at 90o<\/sup>C.\u00a0*We usually do a series of three runs to equal the time\/temp because of evaporation (refill coplin jars with dH2<\/sub>O).<\/li>
    2. Microwave according to instructions on microwave.<\/li>
    3. Cool on countertop, 15 min.<\/li>
    4. Rinse with dH2<\/sub>O<\/li>
    5. 1X PBS, 5 min.<\/li>
    6. Block in 4% Donkey serum\/PBS-T, 2 hours at RT.<\/li>
    7. For Rabbit anti-HOPX<\/strong><\/a> (SC-30216<\/strong><\/a>, Lot # C2212, Santa Cruz) dilute 1:200, For goat anti-SFTPC<\/strong><\/a> (SC-7706, Santa Cruz) dilute 1:100, for mouse anti-\u00a0ACTA2<\/strong><\/a> (A5228, Sigma)\u00a0dilute 1:2000 in blocking buffer. Spin down in \u00b5fuge for 10 min, apply to tissue and incubated O\/N @ 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# 1387792, for anti-HOPX<\/strong><\/a>), Donkey Alexa Fluor 568 anti-goat IgG (A11057<\/strong><\/a>, Lot# 1235787, anti- SFTPC<\/strong><\/a>) at 1:200, Donkey Alexa Fluor 647 anti-mouse IgG ( A31571<\/strong><\/a>, Lot# 1366514, anti- 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. Spin down in \u00b5fuge for 10 min, apply to tissue and incubated at room temperature for 1 hour.<\/li>
      4. Rinse in PBS-T 3X, 5 min.<\/li>
      5. Dilute DAPI 1:2000 and apply to slides for 15 min.<\/li>
      6. Wash in PBS-T 3X, 5 min.<\/li>
      7. Rinse slides in 0.1M PB or TB, 3X, 5 min.<\/li>
      8. Add 1 drop of Prolong Gold anti-fade mounting medium (P36930).<\/li>
      9. Coverslip with Gold Seal Coverslip (Cat# 3422 Electron Microscopy Sciences, 22 X 22 mm).<\/li>
      10. Allow Prolong Gold to cure overnight at room temp in light sealed box.<\/li>
      11. Store slides in a light sealed box @ 4o<\/sup>C.<\/li><\/ol>

        Tissue Used: \u00a0\u00a0 LMM.15.33A.6L.23 <\/strong><\/p>

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

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

        Weight= 6.75g<\/strong><\/p>

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

        Appendix<\/span><\/span><\/p>

        \u00a0<\/span><\/p>

        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>

        pH 6.0<\/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, SFTPC, and ACTA2 \u00a0 Purpose: Stain slides of 5\u00b5m paraffin sections of C57BL\/6J PND14 lungs for HOPX, SFTPC, and ACTA2 with antigen retrieval. Day 1 Paraffin sections are placed at 60oC for 2 hours to overnight to melt paraffin. Paraffin sections are then placed in xylene 3X for 10 minutes each, followed by […]<\/p>\n","protected":false},"author":1,"featured_media":6645,"comment_status":"closed","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"footnotes":""},"categories":[274,107],"tags":[5,6,19,51,29,121],"class_list":["post-6643","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-pro-spc","tag-sftpc","tag-sma"],"yoast_head":"\nPostnatal Day 14 C57BL6 HOPX, SFTPC 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=6643\" \/>\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, SFTPC and ACTA2 Confocal Imaging -\" \/>\n<meta property=\"og:description\" content=\"Immunofluorescence- HOPX, SFTPC, and ACTA2 \u00a0 Purpose: Stain slides of 5\u00b5m paraffin sections of C57BL\/6J PND14 lungs for HOPX, SFTPC, and ACTA2 with antigen retrieval. 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