{"id":32066,"date":"2020-05-21T17:11:37","date_gmt":"2020-05-21T17:11:37","guid":{"rendered":"https:\/\/research.cchmc.org\/lungimage\/?p=32066"},"modified":"2020-05-21T18:15:13","modified_gmt":"2020-05-21T18:15:13","slug":"4-year-old-human-lung-krt14-muc5b-and-tuba4a-confocal-imaging","status":"publish","type":"post","link":"https:\/\/research.cchmc.org\/lungimage\/?p=32066","title":{"rendered":"4 Year-Old Human Lung KRT14, MUC5B, and TUBA4A Confocal Imaging"},"content":{"rendered":"\t\t
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4 Year-Old Human Lung KRT14, MUC5B, and TUBA4A Confocal Imaging<\/h1>\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t
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\"4<\/a>
4 Year Old Human Lung Confocal Imaging Showing Protein Expression of KRT14, MUC5B, and TUBA4A Genes<\/div>
20X Tile Scan<\/div><\/div>\"4<\/a>
4 Year Old Human Lung Confocal Imaging Showing Protein Expression of KRT14, MUC5B, and TUBA4A Genes<\/div>
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4 Year Old Human Lung Confocal Imaging Showing Protein Expression of KRT14, MUC5B, and TUBA4A Genes<\/div>
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4 Year Old Human Lung Confocal Imaging Showing Protein Expression of KRT14, MUC5B, and TUBA4A Genes<\/div>
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4 Year Old Human Lung Confocal Imaging Showing Protein Expression of KRT14, MUC5B, and TUBA4A Genes<\/div>
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4 Year Old Human Lung Confocal Imaging Showing Protein Expression of KRT14, MUC5B, and TUBA4A Genes<\/div>
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4 Year Old Human Lung Confocal Imaging Showing Protein Expression of KRT14, MUC5B, and TUBA4A Genes<\/div>
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4 Year Old Human Lung Confocal Imaging Showing Protein Expression of KRT14, MUC5B, and TUBA4A Genes<\/div>
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4 Year Old Human Lung Confocal Imaging Showing Protein Expression of KRT14, MUC5B, and TUBA4A Genes<\/div>
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4 Year Old Human Lung Confocal Imaging Showing Protein Expression of KRT14, MUC5B, and TUBA4A Genes<\/div>
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4 Year Old Human Lung Confocal Imaging Showing Protein Expression of KRT14, MUC5B, and TUBA4A Genes<\/div>
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4 Year Old Human Lung Confocal Imaging Showing Protein Expression of KRT14, MUC5B, and TUBA4A Genes<\/div>
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4 Year Old Human Lung Confocal Imaging Showing Protein Expression of KRT14, MUC5B, and TUBA4A Genes<\/div>
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4 Year Old Human Lung Confocal Imaging Showing Protein Expression of KRT14, MUC5B, and TUBA4A Genes<\/div>
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4 Year Old Human Lung Confocal Imaging Showing Protein Expression of KRT14, MUC5B, and TUBA4A Genes<\/div>
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4 Year Old Human Lung Confocal Imaging Showing Protein Expression of KRT14, MUC5B, and TUBA4A Genes<\/div>
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4 Year Old Human Lung Confocal Imaging Showing Protein Expression of KRT14, MUC5B, and TUBA4A Genes<\/div>
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4 Year Old Human Lung Confocal Imaging Showing Protein Expression of KRT14, MUC5B, and TUBA4A Genes<\/div>
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4 Year Old Human Lung Confocal Imaging Showing Protein Expression of KRT14, MUC5B, and TUBA4A Genes<\/div>
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4 Year Old Human Lung Confocal Imaging Showing Protein Expression of KRT14, MUC5B, and TUBA4A Genes<\/div>
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Donor Tissue Kindly Provided by Dr. Gloria Pryhuber from the University of Rochester Medical Center<\/p>\n

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

Immunofluorescence- <\/span> <\/span>KRT14<\/span><\/a>,<\/span> MUC5B<\/span><\/a>, <\/span>and TUBA4A<\/span><\/a><\/span><\/p>\n

 <\/span><\/p>\n

Purpose:  <\/span>Stain slides of 7\u00b5m frozen slides of human lung from the HTC for KRT14<\/span><\/a>,<\/span> MUC5B<\/span><\/a>, <\/span>and TUBA4A<\/span><\/a> with antigen retrieval.<\/p>\n

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

    \n
  1. Frozen slides are rehydrated 2X in 1X PBS for 5 minutes each, followed by 5 minutes in 4% paraformaldehyde, followed by a wash in 1X PBS for 5 minutes.<\/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. *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 mouse anti-KRT14<\/span><\/a> (AB7800<\/span><\/a>, Abcam<\/span>) dilute 1:100, for rabbit anti-MUC5B<\/span><\/a> (SC-20119<\/span><\/a>, Lot # B1003 Santa Cruz) Dilute 1:100, For mouse anti-TUBA4A<\/span><\/a> (Acetylated Tubulin, T7451, Sigma) dilute 1:1000 in blocking buffer. Spin down in \u00b5fuge for 20 minutes and incubate on tissue overnight @ 4o<\/sup>C.<\/li>\n<\/ol>\n

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

      \n
    1. Rinse slides in PBS-T 3X, 5 min.<\/li>\n
    2. Apply secondary antibody, Goat Alexa Fluor 488 anti-mouse IgG3 (A21151<\/span><\/a>, Lot# 1563682,  for anti-KRT14<\/span><\/a>) at 1:200, Goat Alexa Fluor 555 anti-rabbit IgG (A21428<\/span><\/a>,<\/span> Lot# 1608466 for anti-MUC5B<\/span><\/a>) at 1:200, Goat Alexa Fluor 647 anti-mouse IgG2B (<\/span>A21242<\/span><\/a>, Lot# 1148959, anti-TUBA4A<\/span><\/a>)  at 1:200, in blocking buffer. Spin down in \u00b5fuge for 20 min, apply to tissue and incubated at room temperature for 1 hour.<\/li>\n
    3. Rinse in PBS-T 3X, 5 min.<\/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 slides at 4o<\/sup>C in a light sealed box.<\/li>\n<\/ol>\n

       <\/span><\/p>\n

      Tissue Used:
      <\/span>LMH-16-<\/b><\/span>D007-LLL-5C4.11<\/b><\/span>
      Gender: Male
      <\/span>Age: 4 Years<\/span><\/p>\n

       <\/span><\/p>\n

      Appendix
      <\/span>Antigen Retrieval Solution<\/span><\/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>\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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      Donor Tissue Kindly Provided by Dr. Gloria Pryhuber from the University of Rochester Medical Center Immunofluorescence-  KRT14, MUC5B, and TUBA4A   Purpose:  Stain slides of 7\u00b5m frozen slides of human lung from the HTC for KRT14, MUC5B, and TUBA4A with antigen retrieval. Day 1 Frozen slides are rehydrated 2X in 1X PBS for 5 minutes each, followed by 5 minutes in 4% paraformaldehyde, followed by a […]<\/p>\n","protected":false},"author":1,"featured_media":0,"comment_status":"closed","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"footnotes":""},"categories":[1],"tags":[],"class_list":["post-32066","post","type-post","status-publish","format-standard","hentry","category-uncategorized"],"yoast_head":"\n4 Year-Old Human Lung KRT14, MUC5B, and TUBA4A 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=32066\" \/>\n<meta property=\"og:locale\" content=\"en_US\" \/>\n<meta property=\"og:type\" content=\"article\" \/>\n<meta property=\"og:title\" content=\"4 Year-Old Human Lung KRT14, MUC5B, and TUBA4A Confocal Imaging -\" \/>\n<meta property=\"og:description\" content=\"Donor Tissue Kindly Provided by Dr. Gloria Pryhuber from the University of Rochester Medical Center Immunofluorescence-  KRT14, MUC5B, and TUBA4A   Purpose:  Stain slides of 7\u00b5m frozen slides of human lung from the HTC for KRT14, MUC5B, and TUBA4A with antigen retrieval. Day 1 Frozen slides are rehydrated 2X in 1X PBS for 5 minutes each, followed by 5 minutes in 4% paraformaldehyde, followed by a […]\" \/>\n<meta property=\"og:url\" content=\"https:\/\/research.cchmc.org\/lungimage\/?p=32066\" \/>\n<meta property=\"article:published_time\" content=\"2020-05-21T17:11:37+00:00\" \/>\n<meta property=\"article:modified_time\" content=\"2020-05-21T18:15:13+00:00\" \/>\n<meta property=\"og:image\" content=\"https:\/\/research.cchmc.org\/lungimage\/wp-content\/uploads\/2015\/07\/Pryhuber_Lab.jpg\" \/>\n<meta name=\"author\" content=\"kitc5i\" \/>\n<meta name=\"twitter:card\" content=\"summary_large_image\" \/>\n<meta name=\"twitter:label1\" content=\"Written by\" \/>\n\t<meta name=\"twitter:data1\" content=\"kitc5i\" \/>\n\t<meta name=\"twitter:label2\" content=\"Est. reading time\" \/>\n\t<meta name=\"twitter:data2\" content=\"2 minutes\" \/>\n<script type=\"application\/ld+json\" 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