{"id":27411,"date":"2018-09-19T17:37:26","date_gmt":"2018-09-19T17:37:26","guid":{"rendered":"https:\/\/research.cchmc.org\/lungimage\/?p=27411"},"modified":"2018-10-12T18:14:30","modified_gmt":"2018-10-12T18:14:30","slug":"7-year-old-human-lung-with-asthma-immunofluorescence-and-confocal-imaging-donor-d078-showing-expression-of-fn1-abca3-and-acta2","status":"publish","type":"post","link":"https:\/\/research.cchmc.org\/lungimage\/?p=27411","title":{"rendered":"7 Year-Old Human Lung with Asthma Immunofluorescence and Confocal Imaging Donor D078 Showing Expression of FN1, ABCA3, and ACTA2"},"content":{"rendered":"\t\t
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7 Year-Old Human Lung with Asthma Immunofluorescence and Confocal Imaging Donor D078 Showing Expression of FN1, ABCA3, and ACTA2<\/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 FN1, ABCA3, and ACTA2<\/div>
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7 Year-Old Human Lung with Asthma Immunofluorescence and Confocal Imaging Donor D078 Showing Expression of FN1, ABCA3, and ACTA2<\/div>
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7 Year-Old Human Lung with Asthma Immunofluorescence and Confocal Imaging Donor D078 Showing Expression of FN1, ABCA3, and ACTA2<\/div>
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7 Year-Old Human Lung with Asthma Immunofluorescence and Confocal Imaging Donor D078 Showing Expression of FN1, ABCA3, and ACTA2<\/div>
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7 Year-Old Human Lung with Asthma Immunofluorescence and Confocal Imaging Donor D078 Showing Expression of FN1, ABCA3, and ACTA2<\/div>
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7 Year-Old Human Lung with Asthma Immunofluorescence and Confocal Imaging Donor D078 Showing Expression of FN1, ABCA3, and ACTA2<\/div>
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7 Year-Old Human Lung with Asthma Immunofluorescence and Confocal Imaging Donor D078 Showing Expression of FN1, ABCA3, and ACTA2<\/div>
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7 Year-Old Human Lung with Asthma Immunofluorescence and Confocal Imaging Donor D078 Showing Expression of FN1, ABCA3, and ACTA2<\/div>
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7 Year-Old Human Lung with Asthma Immunofluorescence and Confocal Imaging Donor D078 Showing Expression of FN1, ABCA3, and ACTA2<\/div>
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7 Year-Old Human Lung with Asthma Immunofluorescence and Confocal Imaging Donor D078 Showing Expression of FN1, ABCA3, and ACTA2<\/div>
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7 Year-Old Human Lung with Asthma Immunofluorescence and Confocal Imaging Donor D078 Showing Expression of FN1, ABCA3, and ACTA2<\/div>
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7 Year-Old Human Lung with Asthma Immunofluorescence and Confocal Imaging Donor D078 Showing Expression of FN1, ABCA3, and ACTA2<\/div>
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7 Year-Old Human Lung with Asthma Immunofluorescence and Confocal Imaging Donor D078 Showing Expression of FN1, ABCA3, and ACTA2<\/div>
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7 Year-Old Human Lung with Asthma Immunofluorescence and Confocal Imaging Donor D078 Showing Expression of FN1, ABCA3, and ACTA2<\/div>
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7 Year-Old Human Lung with Asthma Immunofluorescence and Confocal Imaging Donor D078 Showing Expression of FN1, ABCA3, and ACTA2<\/div>
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7 Year-Old Human Lung with Asthma Immunofluorescence and Confocal Imaging Donor D078 Showing Expression of FN1, ABCA3, and ACTA2<\/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-\u00a0<\/b>FN1<\/b><\/a>,\u00a0<\/b>ABCA3<\/b><\/a>, and\u00a0<\/b>ACTA2<\/b><\/a><\/span><\/p>

\u00a0<\/p>

\u00a0<\/b><\/p>

Purpose:\u00a0\u00a0<\/b>Stain slides of 7\u00b5m frozen sections of 3 year-old human lung provided by the HTC for\u00a0<\/b>FN1<\/b><\/a>, ABCA3, and\u00a0<\/b>ACTA2<\/b><\/a>\u00a0<\/b>with antigen retrieval.<\/b><\/p>

Day 1<\/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% Donkey serum\/PBS-T, 2 hours at RT.<\/li>
  10. For rabbit\u00a0anti-<\/b>FN1<\/b><\/a>\u00a0(AB2413<\/b><\/a>, ABCAM, anti-Fibronectin<\/b><\/a>), for mouse anti-ABCA3<\/b><\/a>\u00a0(WMAB-17G524<\/b><\/a>, Seven Hills Bioreagents) dilute 1:100, for mouse anti-ACTA2<\/b><\/a>\u00a0<\/b>(\u03b1 Smooth muscle actin<\/b><\/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><\/ol>

    Day 2<\/p>

    1. Rinse slides in PBS-T 3X, 5 min.<\/li>
    2. Apply secondary antibody, donkey Alexa Fluor 488 anti-rabbit IgG (A21206<\/b><\/a>, Lot# 1608521, anti-FN1<\/b><\/a>)<\/b>, Goat Alexa Fluor 568 anti-mouse pig IgG2B (A21144<\/b><\/a>, Lot# 1584300, for anti-ABCA3<\/b><\/a>) at 1:200, Donkey Alexa Fluor 633 anti-mouse IgG2A\u00a0A21136<\/b><\/a>, Lot# 1345055,\u00a0ACTA2<\/b>)<\/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>
    3. Wash 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.\u00a0<\/li>
    10. Store slides in light sealed box @ 4o<\/sup>C.<\/li><\/ol>

      Tissue Used: \u00a0\u00a0\u00a0<\/b><\/p>

      LMH-D078-LLL-13B4.18
      Gender: Female
      Age: 7 Years<\/b><\/p>

      \u00a0<\/p>

      \u00a0<\/b><\/p>

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

      \u00a0<\/p>

      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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