{"id":20453,"date":"2018-03-23T18:07:36","date_gmt":"2018-03-23T18:07:36","guid":{"rendered":"https:\/\/research.cchmc.org\/lungimage\/?p=20453"},"modified":"2018-08-14T19:09:28","modified_gmt":"2018-08-14T19:09:28","slug":"pnd01-nkx2-1-abca3-and-acta2-confocal-imaging","status":"publish","type":"post","link":"https:\/\/research.cchmc.org\/lungimage\/?p=20453","title":{"rendered":"PND01 NKX2.1, ABCA3, and ACTA2 Confocal Imaging"},"content":{"rendered":"

\"PND01<\/a>
PND01 Immunofluorescence and Confocal Imaging Showing Expression of NKX2.1, ABCA3, and ACTA2<\/div>
20X<\/div><\/div>\"PND01<\/a>
PND01 Immunofluorescence and Confocal Imaging Showing Expression of NKX2.1, ABCA3, and ACTA2<\/div>
60X<\/div><\/div>\"PND01<\/a>
PND01 Immunofluorescence and Confocal Imaging Showing Expression of NKX2.1, ABCA3, and ACTA2<\/div>
60X<\/div><\/div>\"PND01<\/a>
PND01 Immunofluorescence and Confocal Imaging Showing Expression of NKX2.1, ABCA3, and ACTA2<\/div>
20X<\/div><\/div>\"PND01<\/a>
PND01 Immunofluorescence and Confocal Imaging Showing Expression of NKX2.1, ABCA3, and ACTA2<\/div>
60X<\/div><\/div>\"PND01<\/a>
PND01 Immunofluorescence and Confocal Imaging Showing Expression of NKX2.1, ABCA3, and ACTA2<\/div>
60X<\/div><\/div>\"PND01<\/a>
PND01 Immunofluorescence and Confocal Imaging Showing Expression of NKX2.1, ABCA3, and ACTA2<\/div>
60X<\/div><\/div>\"PND01<\/a>
PND01 Immunofluorescence and Confocal Imaging Showing Expression of NKX2.1, ABCA3, and ACTA2<\/div>
20X<\/div><\/div>\"PND01<\/a>
PND01 Immunofluorescence and Confocal Imaging Showing Expression of NKX2.1, ABCA3, and ACTA2<\/div>
60X<\/div><\/div>\"PND01<\/a>
PND01 Immunofluorescence and Confocal Imaging Showing Expression of NKX2.1, ABCA3, and ACTA2<\/div>
60X<\/div><\/div>\"PND01<\/a>
PND01 Immunofluorescence and Confocal Imaging Showing Expression of NKX2.1, ABCA3, and ACTA2<\/div>
20X<\/div><\/div>\"PND01<\/a>
PND01 Immunofluorescence and Confocal Imaging Showing Expression of NKX2.1, ABCA3, and ACTA2<\/div>
60X<\/div><\/div>\"PND01<\/a>
PND01 Immunofluorescence and Confocal Imaging Showing Expression of NKX2.1, ABCA3, and ACTA2<\/div>
60X<\/div><\/div><\/div><\/div>\n<\/div>
<\/div><\/div><\/div><\/div><\/div>
\n\n\n\n\n\n
Gene
\n<\/span><\/strong><\/td>\n		<\/td>\n<\/td>\n<\/td>\n<\/td>\n<\/td>\n<\/tr>\n
Nkx2.1<\/strong> <\/span><\/td>\nLungGens<\/strong><\/span><\/td>\nEnsemble<\/a><\/strong><\/span><\/td>\nGenecards<\/strong><\/a><\/span><\/td>\nNCBI<\/strong><\/a><\/span><\/td>\nThe Human Protein Atlas<\/strong><\/a><\/span><\/td>\n<\/tr>\n
FOXF1<\/strong><\/span><\/td>\nLungGens<\/strong><\/span><\/td>\nEnsemble<\/strong><\/a><\/span><\/td>\n Genecards<\/strong><\/a> <\/span><\/td>\nNCBI<\/strong><\/a> <\/span><\/td>\nThe Human Protein Atlas<\/strong><\/a><\/span><\/td>\n<\/tr>\n
Acta2<\/strong><\/a><\/span><\/td>\nLungGens<\/strong><\/span><\/td>\nEnsemble<\/strong><\/a><\/span><\/td>\nGenecards<\/strong><\/a> <\/span><\/td>\n NCBI<\/strong><\/a> <\/span><\/td>\nThe Human Protein Atlas<\/strong><\/a><\/span><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Immunofluorescence- NKX2.1, ABCA3, <\/strong>and ACTA2<\/strong><\/a><\/p>\n

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

Purpose:\u00a0 <\/strong>Immunofluorescence on slides of 7\u00b5m frozen sections of C57BL6 PND01 lungs for NKX2.1, ABCA3, <\/strong>and ACTA2<\/strong><\/a> with antigen retrieval.<\/p>\n

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

    \n
  1. For Frozen tissue, rinse 2X in PBS, then 5 min in 4% PFA\/PBS, then rinse 1X in PBS.<\/li>\n
  2. Briefly equilibrate slides in Antigen Retrieval Buffer.<\/li>\n
  3. Antigen retrieval, pH 6.0 (times will vary according to microwave).<\/li>\n
  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>\n
  5. Microwave according to instructions on microwave.<\/li>\n
  6. Cool on countertop, 15 min.<\/li>\n
  7. Rinse with dH2<\/sub>O<\/li>\n
  8. 1X PBS, 5 min.<\/li>\n
  9. Block in 4% Goat serum\/PBS-T, 2 hours at RT.<\/li>\n
  10. For rabbit anti-NKX2.1<\/strong> (RB TTF-1 1231, Lot# 0912A) dilute 1:1000, for guinea pig anti-ABCA3<\/strong> (G985, BL 3\/10\/05, Seven Hills) 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>\n<\/ol>\n

    Day 2 <\/strong><\/p>\n

      \n
    1. Rinse slides in PBS-T 3X, 5 min.<\/li>\n
    2. Apply secondary antibody, Goat Alexa Fluor 488 anti-rabbit IgG (A11034<\/strong><\/a>, Lot# 1298480, for anti- NKX2.1<\/strong>) at 1:200, Goat Alexa Fluor 555 anti-guinea pig IgG (A21435<\/strong><\/a>, Lot# 1571699, for anti-ABCA3<\/strong>) at 1:200, Donkey Alexa Fluor 647 anti-mouse IgG A31571<\/strong><\/a>, Lot# 1549801, 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>\n<\/ol>\n
        \n
      1. Rinse in PBS-T 3X, 5 min.<\/li>\n
      2. Dilute DAPI 1:2000 and apply to slides for 15 min.<\/li>\n
      3. Wash in PBS-T 3X, 5 min.<\/li>\n
      4. Rinse slides in 0.1M PB, 3X, 5 min.<\/li>\n
      5. Add 1 drop of Prolong Gold anti-fade mounting medium (P36930).<\/li>\n
      6. Coverslip with Gold Seal Coverslip (Cat# 3422 Electron Microscopy Sciences, 22 X 22 mm).<\/li>\n
      7. Allow Prolong Gold to cure overnight at room temp in light sealed box.<\/li>\n
      8. Store slides in light sealed box @ 4o<\/sup>C<\/li>\n<\/ol>\n

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

        LMM.15.66A.6.50<\/strong><\/p>\n

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

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

        Weight= 1.14g<\/strong><\/p>\n

        Appendix<\/strong><\/p>\n

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

        Antigen Retrieval Solution<\/strong><\/p>\n

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

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

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

        pH 6.0<\/p>\n<\/div>

        <\/div><\/div><\/div><\/div><\/div>
        <\/div>
        <\/div><\/div><\/div><\/div><\/div>
        <\/div>
        <\/div><\/div><\/div><\/div><\/div>
        <\/div>
        <\/div><\/div><\/div><\/div><\/div><\/p>\n","protected":false},"excerpt":{"rendered":"","protected":false},"author":1,"featured_media":20454,"comment_status":"closed","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"footnotes":""},"categories":[274,106],"tags":[68,5,22],"class_list":["post-20453","post","type-post","status-publish","format-standard","has-post-thumbnail","hentry","category-c57bl6","category-p01-confocal","tag-abca3","tag-acta2","tag-nkx2-1"],"yoast_head":"\nPND01 NKX2.1, ABCA3, 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=20453\" \/>\n<meta property=\"og:locale\" content=\"en_US\" \/>\n<meta property=\"og:type\" content=\"article\" \/>\n<meta property=\"og:title\" content=\"PND01 NKX2.1, ABCA3, and ACTA2 Confocal Imaging -\" \/>\n<meta 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