{"id":13851,"date":"2017-04-12T14:51:55","date_gmt":"2017-04-12T14:51:55","guid":{"rendered":"https:\/\/research.cchmc.org\/lungimage\/?p=13851"},"modified":"2017-04-12T14:51:55","modified_gmt":"2017-04-12T14:51:55","slug":"2-year-human-lung-nkx2-1-ki67-and-acta2-confocal-imaging","status":"publish","type":"post","link":"https:\/\/research.cchmc.org\/lungimage\/?p=13851","title":{"rendered":"2 Year Human Lung NKX2.1, KI67, and ACTA2 Confocal Imaging"},"content":{"rendered":"
\"2<\/a>
2 Year-Old Human Lung Immunofluorescence and Confocal Imaging Showing Expression of Nkx2.1, Ki67, and Acta2 Genes<\/div>
20X<\/div><\/div>\"2<\/a>
2 Year-Old Human Lung Immunofluorescence and Confocal Imaging Showing Expression of Nkx2.1, Ki67, and Acta2 Genes<\/div>
60X<\/div><\/div>\"2<\/a>
2 Year-Old Human Lung Immunofluorescence and Confocal Imaging Showing Expression of Nkx2.1, Ki67, and Acta2 Genes<\/div>
60X<\/div><\/div>\"2<\/a>
2 Year-Old Human Lung Immunofluorescence and Confocal Imaging Showing Expression of Nkx2.1, Ki67, and Acta2 Genes<\/div>
20X<\/div><\/div>\"2<\/a>
2 Year-Old Human Lung Immunofluorescence and Confocal Imaging Showing Expression of Nkx2.1, Ki67, and Acta2 Genes<\/div>
60X<\/div><\/div>\"2<\/a>
2 Year-Old Human Lung Immunofluorescence and Confocal Imaging Showing Expression of Nkx2.1, Ki67, and Acta2 Genes<\/div>
60X<\/div><\/div>\"2<\/a>
2 Year-Old Human Lung Immunofluorescence and Confocal Imaging Showing Expression of Nkx2.1, Ki67, and Acta2 Genes<\/div>
20X<\/div><\/div>\"2<\/a>
2 Year-Old Human Lung Immunofluorescence and Confocal Imaging Showing Expression of Nkx2.1, Ki67, and Acta2 Genes<\/div>
60X<\/div><\/div>\"2<\/a>
2 Year-Old Human Lung Immunofluorescence and Confocal Imaging Showing Expression of Nkx2.1, Ki67, and Acta2 Genes<\/div>
60X<\/div><\/div>\"2<\/a>
2 Year-Old Human Lung Immunofluorescence and Confocal Imaging Showing Expression of Nkx2.1, Ki67, and Acta2 Genes<\/div>
20X<\/div><\/div>\"2<\/a>
2 Year-Old Human Lung Immunofluorescence and Confocal Imaging Showing Expression of Nkx2.1, Ki67, and Acta2 Genes<\/div>
60X<\/div><\/div>\"2<\/a>
2 Year-Old Human Lung Immunofluorescence and Confocal Imaging Showing Expression of Nkx2.1, Ki67, and Acta2 Genes<\/div>
60X<\/div><\/div>\"2<\/a>
2 Year-Old Human Lung Immunofluorescence and Confocal Imaging Showing Expression of Nkx2.1, Ki67, and Acta2 Genes<\/div>
20X<\/div><\/div>\"2<\/a>
2 Year-Old Human Lung Immunofluorescence and Confocal Imaging Showing Expression of Nkx2.1, Ki67, and Acta2 Genes<\/div>
60X<\/div><\/div>\"2<\/a>
2 Year-Old Human Lung Immunofluorescence and Confocal Imaging Showing Expression of Nkx2.1, Ki67, and Acta2 Genes<\/div>
60X<\/div><\/div>\"2<\/a>
2 Year-Old Human Lung Immunofluorescence and Confocal Imaging Showing Expression of Nkx2.1, Ki67, and Acta2 Genes<\/div>
20X<\/div><\/div>\"2<\/a>
2 Year-Old Human Lung Immunofluorescence and Confocal Imaging Showing Expression of Nkx2.1, Ki67, and Acta2 Genes<\/div>
60X<\/div><\/div>\"2<\/a>
2 Year-Old Human Lung Immunofluorescence and Confocal Imaging Showing Expression of Nkx2.1, Ki67, and Acta2 Genes<\/div>
60X<\/div><\/div><\/div><\/div>\n<\/div>
<\/div><\/div><\/div>

Donor Tissue Kindly Provided by Dr. Gloria Pryhuber from\u00a0the University of Rochester Medical Center<\/p>\n

\"Pryhuber_Lab\"<\/a><\/p>\n

 <\/p>\n\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
Ki67<\/a><\/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

 <\/p>\n

Immunofluorescence-NKX2.1, <\/strong>Ki-67<\/strong><\/a> and ACTA2<\/strong><\/a><\/span><\/p>\n

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

Purpose: \u00a0<\/strong>Immunofluorescence of 7 \u00b5m frozen section of 2 year-old human lung provided by the HTC for NKX2.1, <\/strong>Ki-67<\/strong><\/a> 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 (RB TTF-1 1231, Lot# 0912A) dilute 1:1000, For mouse anti-Ki-67<\/strong><\/a> (55603<\/strong><\/a>, BD Biosciences, Lot# 6251774) 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,\u00a0 for anti-NKX2.1<\/strong>), Goat Alexa Fluor 568 anti-mouse IgG1 (A21124<\/strong><\/a>, Lot# 1600878, for anti-Ki-67<\/strong><\/a>) at 1:200, Donkey Alexa Fluor 633 anti-mouse IgG2A (A21136<\/strong><\/a>, Lot# 1345055, 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
    3. Rinse in PBS-T 5X.c<\/li>\n
    4. Dilute DAPI 1:2000 and apply to slides for 10 min.<\/li>\n
    5. Wash in PBS-T 3X, 5 min.<\/li>\n
    6. Rinse slides in 0.1M PB or TB, 3X, 5 min.<\/li>\n
    7. Add 1 drop of Prolong Gold anti-fade mounting medium (P36930).<\/li>\n
    8. Coverslip with Gold Seal Coverslip (Cat# 3422 Electron Microscopy Sciences, 22 X 22 mm).<\/li>\n
    9. Allow Prolong Gold to cure overnight at room temp in light sealed box.<\/li>\n
    10. Store in light-sealed box at 4o<\/sup>C.<\/li>\n<\/ol>\n

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

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

      LMH-16-D022-RLL-6B2.13<\/strong><\/p>\n

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

      Age: 2 Years<\/strong><\/p>\n

      Gestational Age at Birth:\u00a0 36 weeks<\/strong><\/p>\n

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

      Appendix<\/strong><\/span><\/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>\n","protected":false},"excerpt":{"rendered":"","protected":false},"author":1,"featured_media":13852,"comment_status":"closed","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"footnotes":""},"categories":[79],"tags":[5,6,163,22,36],"class_list":["post-13851","post","type-post","status-publish","format-standard","has-post-thumbnail","hentry","category-perinatalhuman","tag-acta2","tag-alpha-smooth-muscle-actin","tag-ki67","tag-nkx2-1","tag-ttf-1"],"yoast_head":"\n2 Year Human Lung NKX2.1, KI67, 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=13851\" \/>\n<meta property=\"og:locale\" content=\"en_US\" \/>\n<meta property=\"og:type\" content=\"article\" \/>\n<meta property=\"og:title\" content=\"2 Year Human Lung NKX2.1, KI67, and ACTA2 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