{"id":8688,"date":"2016-07-05T18:15:43","date_gmt":"2016-07-05T18:15:43","guid":{"rendered":"https:\/\/research.cchmc.org\/lungimage\/?p=8688"},"modified":"2018-11-12T18:31:50","modified_gmt":"2018-11-12T18:31:50","slug":"3-year-old-human-lung-krt14-muc5b-tuba4a-confocal-imaging","status":"publish","type":"post","link":"https:\/\/research.cchmc.org\/lungimage\/?p=8688","title":{"rendered":"3 Year-Old Human Lung KRT14, MUC5B, TUBA4A Confocal Imaging"},"content":{"rendered":"\t\t
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3 Year-Old Human Lung KRT14, MUC5B, TUBA4A Confocal Imaging<\/h1>\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t
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3 Year Old Human Lung Confocal Imaging Showing Protein Expression of KRT14, MUC5B, and TUBA4A Genes<\/div>
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3 Year Old Human Lung Confocal Imaging Showing Protein Expression of KRT14, MUC5B, and TUBA4A Genes<\/div>
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3 Year Old Human Lung Confocal Imaging Showing Protein Expression of KRT14, MUC5B, and TUBA4A Genes<\/div>
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3 Year Old Human Lung Confocal Imaging Showing Protein Expression of KRT14, MUC5B, and TUBA4A Genes<\/div>
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3 Year Old Human Lung Confocal Imaging Showing Protein Expression of KRT14, MUC5B, and TUBA4A Genes<\/div>
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3 Year Old Human Lung Confocal Imaging Showing Protein Expression of KRT14, MUC5B, and TUBA4A Genes<\/div>
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3 Year Old Human Lung Confocal Imaging Showing Protein Expression of KRT14, MUC5B, and TUBA4A Genes<\/div>
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3 Year Old Human Lung Confocal Imaging Showing Protein Expression of KRT14, MUC5B, and TUBA4A Genes<\/div>
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3 Year Old Human Lung Confocal Imaging Showing Protein Expression of KRT14, MUC5B, and TUBA4A Genes<\/div>
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3 Year Old Human Lung Confocal Imaging Showing Protein Expression of KRT14, MUC5B, and TUBA4A Genes<\/div>
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3 Year Old Human Lung Confocal Imaging Showing Protein Expression of KRT14, MUC5B, and TUBA4A Genes<\/div>
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60X<\/div><\/div><\/div><\/div><\/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\u00a0the University of Rochester Medical Center<\/p>

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

\u00a0<\/p>

Immunofluorescence-\u00a0<\/span>\u00a0<\/span>KRT14<\/span><\/a>,<\/span>\u00a0MUC5B<\/span><\/a>,\u00a0<\/span>and\u00a0TUBA4A<\/span><\/a><\/span><\/p>

\u00a0<\/span><\/p>

Purpose:\u00a0\u00a0<\/span>Stain slides of 5\u00b5m paraffin slides of human lung from the HTC for\u00a0KRT14<\/span><\/a>,<\/span>\u00a0MUC5B<\/span><\/a>,\u00a0<\/span>and\u00a0TUBA4A<\/span><\/a>\u00a0with antigen retrieval.<\/p>

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

  1. Paraffin sections are placed O\/N at 60o<\/sup>C to melt paraffin.<\/li>
  2. 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>
  3. Briefly equilibrate slides in Antigen Retrieval Buffer.<\/li>
  4. Antigen retrieval, pH 6.0 (times will vary according to microwave).<\/li>
  5. 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>
  6. Microwave according to instructions on microwave.<\/li>
  7. Cool on countertop, 15 min.<\/li>
  8. Rinse with dH2<\/sub>O<\/li>
  9. 1X PBS, 5 min.<\/li>
  10. Block in 4% Goat serum\/PBS-T, 2 hours at RT.<\/li>
  11. For mouse anti-KRT14<\/span><\/a>\u00a0(AB7800<\/span><\/a>, Abcam<\/span>) dilute 1:100, for rabbit anti-MUC5B<\/span><\/a>\u00a0(SC-20119<\/span><\/a>, Lot # B1003 Santa Cruz) Dilute 1:100, For mouse anti-TUBA4A<\/span><\/a>\u00a0(Acetylated Tubulin, T7451, Sigma) dilute 1:1000 in blocking buffer. Spin down in \u00b5fuge for 10 minutes and incubate on tissue overnight @ 4o<\/sup>C.<\/li><\/ol>

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

    1. Rinse slides in PBS-T 3X, 5 min.<\/li>
    2. Apply secondary antibody, Goat Alexa Fluor 488 anti-mouse IgG3 (A21151<\/span><\/a>, Lot# 1563682,\u00a0 for anti-KRT14<\/span><\/a>) at 1:200, Goat Alexa Fluor 555 anti-rabbit IgG (A21428<\/span><\/a>,<\/span>\u00a0Lot# 1608466 for anti-MUC5B<\/span><\/a>) at 1:200, Goat Alexa Fluor 647 anti-mouse IgG2B\u00a0(<\/span>A21242<\/span><\/a>, Lot# 1148959, anti-TUBA4A<\/span><\/a>)\u00a0 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>
    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>
    10. Store slides at 4o<\/sup>C in a light sealed box.<\/li><\/ol>

      \u00a0<\/span><\/p>

      Tissue Used:
      <\/span>LMH-16-D032-RLL-9B1.4
      <\/span>LMH-16-D032-RLL-10B2.3
      <\/span>Gender: Male
      <\/span>Age: 3 Years<\/span><\/p>

      \u00a0<\/p>

      \u00a0<\/span><\/p>

      Appendix
      <\/span>Antigen Retrieval Solution<\/span><\/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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