{"id":28898,"date":"2018-11-06T13:19:38","date_gmt":"2018-11-06T13:19:38","guid":{"rendered":"https:\/\/research.cchmc.org\/lungimage\/?p=28898"},"modified":"2018-11-13T17:48:24","modified_gmt":"2018-11-13T17:48:24","slug":"7-year-old-human-lung-with-asthma-immunofluorescence-and-confocal-imaging-donor-d078-showing-expression-of-krt14-sox2-and-muc5b","status":"publish","type":"post","link":"https:\/\/research.cchmc.org\/lungimage\/?p=28898","title":{"rendered":"7 Year-Old Human Lung with Asthma Immunofluorescence and Confocal Imaging Donor D078 Showing Expression of KRT14, SOX2, and MUC5B"},"content":{"rendered":"\t\t
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7 Year-Old Human Lung with Asthma Immunofluorescence and Confocal Imaging Donor D078 Showing Expression of KRT14, SOX2, and MUC5B<\/h1>\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t
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\"7<\/a>
7 Year-Old Human Lung with Asthma Immunofluorescence and Confocal Imaging Donor D078 Showing Expression of KRT14, SOX2, and MUC5B<\/div>
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7 Year-Old Human Lung with Asthma Immunofluorescence and Confocal Imaging Donor D078 Showing Expression of KRT14, SOX2, and MUC5B<\/div>
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7 Year-Old Human Lung with Asthma Immunofluorescence and Confocal Imaging Donor D078 Showing Expression of KRT14, SOX2, and MUC5B<\/div>
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7 Year-Old Human Lung with Asthma Immunofluorescence and Confocal Imaging Donor D078 Showing Expression of KRT14, SOX2, and MUC5B<\/div>
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7 Year-Old Human Lung with Asthma Immunofluorescence and Confocal Imaging Donor D078 Showing Expression of KRT14, SOX2, and MUC5B<\/div>
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7 Year-Old Human Lung with Asthma Immunofluorescence and Confocal Imaging Donor D078 Showing Expression of KRT14, SOX2, and MUC5B<\/div>
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7 Year-Old Human Lung with Asthma Immunofluorescence and Confocal Imaging Donor D078 Showing Expression of KRT14, SOX2, and MUC5B<\/div>
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7 Year-Old Human Lung with Asthma Immunofluorescence and Confocal Imaging Donor D078 Showing Expression of KRT14, SOX2, and MUC5B<\/div>
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7 Year-Old Human Lung with Asthma Immunofluorescence and Confocal Imaging Donor D078 Showing Expression of KRT14, SOX2, and MUC5B<\/div>
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7 Year-Old Human Lung with Asthma Immunofluorescence and Confocal Imaging Donor D078 Showing Expression of KRT14, SOX2, and MUC5B<\/div>
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7 Year-Old Human Lung with Asthma Immunofluorescence and Confocal Imaging Donor D078 Showing Expression of KRT14, SOX2, and MUC5B<\/div>
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7 Year-Old Human Lung with Asthma Immunofluorescence and Confocal Imaging Donor D078 Showing Expression of KRT14, SOX2, and MUC5B<\/div>
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7 Year-Old Human Lung with Asthma Immunofluorescence and Confocal Imaging Donor D078 Showing Expression of KRT14, SOX2, and MUC5B<\/div>
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7 Year-Old Human Lung with Asthma Immunofluorescence and Confocal Imaging Donor D078 Showing Expression of KRT14, SOX2, and MUC5B<\/div>
60X<\/div><\/div>\"7<\/a>
7 Year-Old Human Lung with Asthma Immunofluorescence and Confocal Imaging Donor D078 Showing Expression of KRT14, SOX2, and MUC5B<\/div>
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\u00a0\u00a0Donor Tissue Kindly Provided by Dr. Gloria Pryhuber from\u00a0the University of Rochester Medical Center<\/p>

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

Immunofluorescence-<\/strong>KRT14<\/strong><\/a>, <\/strong>SOX2<\/strong><\/a> and MUC5B<\/strong><\/a><\/span><\/p>

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

Purpose: \u00a0<\/strong>Immunofluorescence of 7\u00b5m frozen sections of lungs provided by HTC for KRT14<\/strong><\/a>, <\/strong>SOX2<\/strong><\/a> and MUC5B<\/strong><\/a> with antigen retrieval.<\/p>

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

  1. For Frozen tissue, rinse 2X in PBS, then 5 min in 4% PFA\/PBS, then rinse 1X in PBS.<\/li>
  2. Briefly equilibrate slides in Antigen Retrieval Buffer.<\/li>
  3. Antigen retrieval, pH 6.0 (times will vary according to microwave).<\/li>
  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>
  5. Microwave according to instructions on microwave.<\/li>
  6. Cool on countertop, 15 min.<\/li>
  7. Rinse with dH2<\/sub>O<\/li>
  8. 1X PBS, 5 min.<\/li>
  9. Block in 4% Goat serum\/PBS-T, 2 hours at RT.<\/li>
  10. For mouse anti-KRT14<\/strong><\/a> (AB7800<\/strong><\/a>, Abcam<\/strong>) dilute 1:100, For mouse anti-SOX2<\/strong><\/a> (SC-365823<\/strong><\/a>, Santa Cruz, Lot# C0317) dilute 1:100, for rabbit anti-MUC5B<\/strong><\/a>\u00a0(SC-20119<\/strong><\/a>, Lot # B1003 Santa Cruz) Dilute 1:100 in blocking buffer. Spin down in \u00b5fuge for 10 minutes 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-mouse IgG3 (A21151<\/strong><\/a>, Lot# 1563682,\u00a0 for anti-KRT14<\/strong><\/a>) at 1:200, Goat Alexa Fluor 568 anti-mouse IgG1 (A21124<\/strong><\/a>, Lot# 1600878, for anti-SOX2<\/strong><\/a>) at 1:200, Goat Alexa Fluor 633 anti-rabbit IgG (A21070<\/strong><\/a>, Lot # 1668691, anti-MUC5B<\/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>
    3. Rinse in PBS-T 5X<\/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 in light-sealed box at 4o<\/sup>C.<\/li><\/ol>

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

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

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

      Tissue Used:<\/b>
      2018-11-05_LMH-D078-LLL-12B5.20
      Age: 7 Years
      Gender: Female\u00a0<\/b><\/p>

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

      Appendix
      <\/strong>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>

      pH 6.0<\/p>

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