{"id":2035,"date":"2015-04-22T00:53:54","date_gmt":"2015-04-22T00:53:54","guid":{"rendered":"https:\/\/research.cchmc.org\/lungimage\/?p=2035"},"modified":"2018-08-17T22:55:33","modified_gmt":"2018-08-17T22:55:33","slug":"e18-5-c57bl6-hopx-sftpc-and-acta2-confocal-imaging","status":"publish","type":"post","link":"https:\/\/research.cchmc.org\/lungimage\/?p=2035","title":{"rendered":"E18.5 C57BL6 HOPX, SFTPC, and ACTA2 Confocal Imaging"},"content":{"rendered":"\t\t
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E18.5 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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\"E18.5<\/a>
E18.5 Confocal Imaging Showing Protein Expression of Hopx, Sftpc, and Acta2 Genes<\/div>
20X Tile Scan<\/div><\/div>\"E18.5<\/a>
E18.5 Confocal Imaging Showing Protein Expression of Hopx, Sftpc, and Acta2 Genes<\/div>
20X<\/div><\/div>\"2015-001-008_C57Bl6_E16.5_LMM.14.57.4.39_Hopx_SPC_Acta2\"<\/a>
2015-001-008_C57Bl6_E16.5_LMM.14.57.4.39_Hopx_SPC_Acta2<\/div>
60X<\/div><\/div>\"E18.5<\/a>
E18.5 Confocal Imaging Showing Protein Expression of Hopx, Sftpc, and Acta2 Genes<\/div>
20X<\/div><\/div>\"E18.5<\/a>
E18.5 Confocal Imaging Showing Protein Expression of Hopx, Sftpc, and Acta2 Genes<\/div>
60X<\/div><\/div>\"E18.5<\/a>
E18.5 Confocal Imaging Showing Protein Expression of Hopx, Sftpc, and Acta2 Genes<\/div>
20X<\/div><\/div>\"E18.5<\/a>
E18.5 Confocal Imaging Showing Protein Expression of Hopx, Sftpc, and Acta2 Genes<\/div>
60X<\/div><\/div>\"E18.5<\/a>
E18.5 Confocal Imaging Showing Protein Expression of Hopx, Sftpc, and Acta2 Genes<\/div>
20X<\/div><\/div>\"E18.5<\/a>
E18.5 Confocal Imaging Showing Protein Expression of Hopx, Sftpc, and Acta2 Genes<\/div>
60X<\/div><\/div><\/div><\/div><\/div>\n\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- <\/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 E18.5 lungs for HOPX<\/strong><\/a>,<\/strong> SFTPC<\/strong><\/a>, <\/strong>and ACTA2<\/strong><\/a> with antigen retrieval.<\/p><\/div>

Day 1<\/b><\/span><\/div>
\u00a0<\/div>
  1. Paraffin sections are placed at 60 o<\/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.<\/span><\/li>
  2. For Frozen tissue remove OCT and rehydrate by washing in 2X in PBS, then 5 min in 4% PFA\/PBS, then rinse 1X in PBS.<\/span><\/li>
  3. Briefly equilibrate slides in Antigen Retrieval Buffer.<\/span><\/li>
  4. 10 mM sodium citrate, pH 6.0, and heat in a microwave for 15 min at 96 o<\/sup>C. *We usually do a series of three runs to equal the time\/temp because of evaporation (refill coplin jars with dH2<\/sub>O).<\/span><\/li>
  5. Microwave according to instructions on microwave.<\/span><\/li>
  6. Cool on countertop, 15 min.<\/span><\/li>
  7. Rinse with dH 2<\/sub>O<\/span><\/li>
  8. 1X PBS, 5 min.<\/span><\/li>
  9. Block in 4% Donkey serum\/PBS-T, 2 hours at RT.<\/span><\/li>
  10. For Rabbit anti-<\/span> HOPX<\/span><\/span><\/a> <\/strong>(<\/span>SC-30216<\/span><\/span><\/a><\/strong>, Lot # C2212, Santa Cruz<\/span>) dilute 1:200, For goat anti-<\/span>SFTPC<\/span><\/span><\/a><\/strong> (SC-7706, Santa Cruz) dilute 1:100,<\/span> for<\/span> mouse anti- <\/span> ACTA2<\/span><\/b><\/span><\/a> (A5228, Sigma) dilute 1:2000 in blocking buffer. Spin down in \u00b5fuge for 10 min, apply to tissue and<\/span> incubated O\/N @ 4o<\/sup> C.<\/span><\/li><\/ol>
    Day 2<\/b><\/span><\/div>
    \u00a0<\/div>
    1. Rinse slides in PBS-T 3X, 5 min.<\/span><\/li>
    2. Apply secondary antibody , Donkey Alexa Fluor 488 anti-rabbit IgG (<\/span> A21206<\/span><\/span><\/a><\/strong>, Lot# 1387792, for anti-<\/span>HOPX<\/span><\/span><\/a><\/strong>), Donkey Alexa Fluor 568 anti-goat IgG (<\/span> A11057<\/span><\/span><\/a><\/strong>, Lot# 1235787, anti-<\/span> SFTPC<\/span><\/span><\/a><\/strong>) at 1:200, Donkey Alexa Fluor 647 anti-mouse IgG<\/span> A31571<\/span><\/span><\/a><\/strong>, Lot# 1366514, anti-<\/span> ACTA2<\/span><\/b><\/span><\/a>) at 1:200, in blocking buffer. Spin down in \u00b5fuge for 10 min, apply to tissue and<\/span> incubated at<\/span> room temperature for 1 hour<\/span>.<\/span><\/li>
    3. Rinse in PBS-T 3X, 5 min.<\/li>
    4. Dilute DAPI 1:2000 and apply to slides for 15 min.<\/span><\/li>
    5. Wash in PBS-T 3X, 5 min.<\/span><\/li>
    6. Rinse slides in 0.1M PB or TB, 3X, 5 min.<\/span><\/li>
    7. Add 1 drop of Prolong Gold anti-fade mounting medium (P36930).<\/span><\/li>
    8. Coverslip with Gold Seal Coverslip (Cat# 3422 Electron Microscopy Sciences, 22 X 22 mm).<\/span><\/li>
    9. Allow Prolong Gold to cure overnight at room temp in light sealed box.<\/span><\/li>
    10. Store slides in a light sealed box\u00a0<\/span>@ 4o<\/sup>\u00a0C.<\/span><\/li><\/ol>
      \u00a0<\/div>
      Tissue Used: <\/b><\/span><\/div>

      LMM.14.49.5.55 E18.5<\/p>

      Gender: Male<\/p>

      Crown-to-Rump Length: 22.0 mm<\/p>

      Weight=1.17g<\/p><\/div>

      \u00a0<\/div>
      Appendix<\/b><\/span><\/div>
      \u00a0<\/div>
      Antigen Retrieval Solution<\/b><\/span><\/div>
      18ml Solution A- 0.1M Citric Acid (Sigma, C1909) pH 2.5<\/span><\/div>
      82ml Reagent B- 0.1M Sodium Citrate (Sigma, S4641 ) pH 8.2<\/span><\/div>
      1L dH2<\/sub> O<\/span><\/div>
      pH 6.0<\/span><\/div>
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      Immunofluorescence- HOPX, SFTPC, and ACTA2 \u00a0 Purpose: Stain slides of 5\u00b5m paraffin sections of C57BL\/6J E18.5 lungs for HOPX, SFTPC, and ACTA2 with antigen retrieval. Day 1 Paraffin sections are placed at 60 oC for 2 hours to overnight to melt paraffin. Paraffin sections are then placed in xylene 3X for 10 minutes each, followed […]<\/p>\n","protected":false},"author":1,"featured_media":26047,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"footnotes":""},"categories":[83,274],"tags":[5,6,19,29],"class_list":["post-2035","post","type-post","status-publish","format-standard","has-post-thumbnail","hentry","category-e18-5","category-c57bl6","tag-acta2","tag-alpha-smooth-muscle-actin","tag-hopx","tag-sftpc"],"yoast_head":"\nE18.5 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=2035\" \/>\n<meta property=\"og:locale\" content=\"en_US\" \/>\n<meta property=\"og:type\" content=\"article\" \/>\n<meta property=\"og:title\" content=\"E18.5 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 E18.5 lungs for HOPX, SFTPC, and ACTA2 with antigen retrieval. 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