{"id":607,"date":"2015-02-08T23:52:38","date_gmt":"2015-02-08T23:52:38","guid":{"rendered":"http:\/\/joekitz.com\/LungMap\/?p=607"},"modified":"2018-08-14T16:50:14","modified_gmt":"2018-08-14T16:50:14","slug":"e16-5_c57bl6_hopx_nkx2-1_acta2_confocal-imaging","status":"publish","type":"post","link":"https:\/\/research.cchmc.org\/lungimage\/?p=607","title":{"rendered":"E16.5 C57Bl6 HOPX, NKX2.1, and ACTA2 Confocal Imaging"},"content":{"rendered":"\t\t
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E16.5 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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\"E16.5<\/a>
E16.5 Confocal Imaging Showing Protein Expression of Hopx, Nkx2.1, and Acta2 Genes<\/div>
20X<\/div><\/div>\"E16.5<\/a>
E16.5 Confocal Imaging Showing Protein Expression of Hopx, Nkx2.1, and Acta2 Genes<\/div>
60X<\/div><\/div>\"E16.5<\/a>
E16.5 Confocal Imaging Showing Protein Expression of Hopx, Nkx2.1, and Acta2 Genes<\/div>
20X<\/div><\/div>\"E16.5<\/a>
E16.5 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>

Purpose:<\/strong>\u00a0 <\/strong>Stain slides of paraffin sections of C57BL6 E16.5 lung for <\/span>HOPX<\/strong><\/a>, NKX2.1, and <\/strong>ACTA2<\/strong><\/a> with antigen retrieval.<\/span><\/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>
  3. 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>
  4. Microwave according to instructions on microwave.<\/li>
  5. Cool on countertop, 15 min.<\/li>
  6. Rinse with dH2<\/sub>O.<\/li>
  7. 1X PBS, 5 min.<\/li>
  8. Block in 4% Goat serum\/PBS-T, 2 hours at RT.<\/li>
  9. For Rabbit anti-HOPX<\/strong><\/a> (SC-30216<\/a>, Lot # C2212, Santa Cruz) dilute 1:200, For guinea pig anti-NKX2.1 (G237, Bl. 10\/7\/11) dilute 1:500, for mouse anti-ACTA2<\/strong><\/a>\u00a0(A5228<\/a>, Sigma)\u00a0dilute 1:2000 in blocking buffer 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, for anti-HOPX<\/strong><\/a>), Goat Alexa Fluor 555 anti-guinea pig IgG (A21435<\/strong><\/a>, Lot# 1571699, NKX2.1) at 1:200 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 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><\/ol>

      Tissue Used: \u00a0\u00a0<\/strong><\/p>

      LMM.14.24.4.30 E16.5<\/p>

      Gender: Male<\/p>

      Crown-to-Rump Length: 16.0 mm<\/p>

      Weight=0.592g<\/p>

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

      Appendix<\/strong><\/p>

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

      Antigen Retrieval Solution<\/strong><\/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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      Immunofluorescence- HOPX, NKX2.1, and ACTA2 Purpose:\u00a0 Stain slides of paraffin sections of C57BL6 E16.5 lung for HOPX, NKX2.1, 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 3X in […]<\/p>\n","protected":false},"author":1,"featured_media":1452,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"footnotes":""},"categories":[81,274],"tags":[5,6,19,22,36],"class_list":["post-607","post","type-post","status-publish","format-standard","has-post-thumbnail","hentry","category-e16-5","category-c57bl6","tag-acta2","tag-alpha-smooth-muscle-actin","tag-hopx","tag-nkx2-1","tag-ttf-1"],"yoast_head":"\nE16.5 C57Bl6 HOPX, 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=607\" \/>\n<meta property=\"og:locale\" content=\"en_US\" \/>\n<meta property=\"og:type\" content=\"article\" \/>\n<meta property=\"og:title\" content=\"E16.5 C57Bl6 HOPX, NKX2.1, and ACTA2 Confocal Imaging -\" \/>\n<meta property=\"og:description\" content=\"Immunofluorescence- HOPX, NKX2.1, and ACTA2 Purpose:\u00a0 Stain slides of paraffin sections of C57BL6 E16.5 lung for HOPX, NKX2.1, and ACTA2 with antigen retrieval. 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