{"id":19920,"date":"2018-03-06T19:41:20","date_gmt":"2018-03-06T19:41:20","guid":{"rendered":"https:\/\/research.cchmc.org\/lungimage\/?p=19920"},"modified":"2018-11-12T16:15:46","modified_gmt":"2018-11-12T16:15:46","slug":"2-year-old-human-lung-confocal-imaging-donor-d022-fn1-foxf1-and-acta2","status":"publish","type":"post","link":"https:\/\/research.cchmc.org\/lungimage\/?p=19920","title":{"rendered":"2 Year-Old Human Lung Confocal Imaging Donor D022 FN1, FOXF1, and ACTA2"},"content":{"rendered":"\t\t
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2 Year-Old Human Lung Confocal Imaging Donor D022 FN1, FOXF1, and ACTA2<\/h1>\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t
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\"2<\/a>
2 Year-Old Human Lung Immunofluorescence and Confocal Imaging Donor D022 Showing Expressions of FN1, FOXF1, and ACTA2<\/div>
20X<\/div><\/div>\"2<\/a>
2 Year-Old Human Lung Immunofluorescence and Confocal Imaging Donor D022 Showing Expressions of FN1, FOXF1, and ACTA2<\/div>
60X<\/div><\/div>\"2<\/a>
2 Year-Old Human Lung Immunofluorescence and Confocal Imaging Donor D022 Showing Expressions of FN1, FOXF1, and ACTA2<\/div>
60X<\/div><\/div>\"2<\/a>
2 Year-Old Human Lung Immunofluorescence and Confocal Imaging Donor D022 Showing Expressions of FN1, FOXF1, and ACTA2<\/div>
20X<\/div><\/div>\"2<\/a>
2 Year-Old Human Lung Immunofluorescence and Confocal Imaging Donor D022 Showing Expressions of FN1, FOXF1, and ACTA2<\/div>
60X<\/div><\/div>\"2<\/a>
2 Year-Old Human Lung Immunofluorescence and Confocal Imaging Donor D022 Showing Expressions of FN1, FOXF1, and ACTA2<\/div>
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2 Year-Old Human Lung Immunofluorescence and Confocal Imaging Donor D022 Showing Expressions of FN1, FOXF1, and ACTA2<\/div>
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2 Year-Old Human Lung Immunofluorescence and Confocal Imaging Donor D022 Showing Expressions of FN1, FOXF1, and ACTA2<\/div>
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2 Year-Old Human Lung Immunofluorescence and Confocal Imaging Donor D022 Showing Expressions of FN1, FOXF1, and ACTA2<\/div>
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2 Year-Old Human Lung Immunofluorescence and Confocal Imaging Donor D022 Showing Expressions of FN1, FOXF1, and ACTA2<\/div>
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2 Year-Old Human Lung Immunofluorescence and Confocal Imaging Donor D022 Showing Expressions of FN1, FOXF1, and ACTA2<\/div>
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2 Year-Old Human Lung Immunofluorescence and Confocal Imaging Donor D022 Showing Expressions of FN1, FOXF1, and ACTA2<\/div>
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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 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0\u00a0<\/span><\/strong>Immunofluorescence-\u00a0<\/span><\/strong>FN1<\/strong><\/a>,\u00a0<\/strong>FOXF1<\/strong><\/a>, and\u00a0<\/strong>ACTA2<\/strong><\/a><\/span><\/p>

Purpose:\u00a0\u00a0<\/span><\/b>Stain slides of 7\u00b5m frozen sections of human lung from HTC for\u00a0FN1<\/b><\/a>,\u00a0<\/b>FOXF1<\/b><\/a>, and\u00a0<\/b>ACTA2<\/b><\/a>.<\/span><\/p>

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

  1. For Frozen tissue, rinse 2X in PBS, then 5 min in 4% PFA\/PBS, then rinse 1X in PBS.<\/span><\/li>
  2. Briefly equilibrate slides in Antigen Retrieval Buffer.<\/span><\/li>
  3. Antigen retrieval, pH 6.0 (times will vary according to microwave).<\/span><\/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).<\/span><\/li>
  5. Microwave according to instructions on microwave.<\/span><\/li>
  6. Cool on countertop, 15 min.<\/span><\/li>
  7. Rinse with dH2<\/sub>O<\/span><\/li>
  8. 1X PBS, 5 min.<\/span><\/li>
  9. Block in 4% Donkey serum\/PBS-T, 2 hours at RT.<\/span><\/li>
  10. For Rabbit\u00a0anti-<\/b>FN1<\/b><\/a>\u00a0(AB2413<\/b><\/a>, ABCAM, anti-Fibronectin<\/b><\/a>) dilute 1:100,
    For goat anti-\u00a0    FOXF1<\/b><\/a>\u00a0dilute 1:100, for mouse anti-ACTA2<\/b><\/a>\u00a0(\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.<\/span><\/li><\/ol>

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

    1. Rinse slides in PBS-T 3X, 5 min.<\/span><\/li>
    2. Apply secondary antibody, Donkey Alexa Fluor 488 anti-rabbit IgG (A21206<\/b><\/a>, Lot# 1608521, anti-FN1<\/b><\/a>)<\/b>\u00a0Donkey
      Alexa Fluor 568 anti-goat IgG (      A11057<\/b><\/a>, Lot# 1485187, for anti-FOXF1<\/b><\/a>), at 1:200, Donkey Alexa Fluor 647 anti-mouse
      IgG (      A31571<\/b><\/a>, Lot# 1549801,\u00a0ACTA2<\/b>)<\/a>\u00a0at 1:200, in blocking buffer. Spin down in \u00b5fuge for 10 min, apply to tissue
      and incubated at room temperature for 1 hour.<\/span><\/li>
    3. Wash in PBS-T 3X, 5 min.<\/span><\/li>
    4. Dilute DAPI 1:2000 and apply to slides for 10 min.<\/span><\/li>
    5. Wash in PBS-T 3X, 5 min.<\/span><\/li>
    6. Rinse slides in 0.1M PB or TB, 3X, 5 min.<\/span><\/li>
    7. Add 1 drop of Prolong Gold anti-fade mounting medium (P36930).<\/span><\/li>
    8. Coverslip with Gold Seal Coverslip (Cat# 3422 Electron Microscopy Sciences, 22 X 22 mm).<\/span><\/li>
    9. Allow Prolong Gold to cure overnight at room temp in light sealed box.<\/span><\/li><\/ol>
      1. Store slides in light sealed box @ 4o<\/sup>C<\/span><\/li><\/ol>

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

        Tissue Used:
        <\/span><\/b>LMH-15-D022-LLL-4A4.17
        <\/span><\/b>Age: 2 Years
        <\/span><\/b>Gender: Male<\/span><\/b><\/p>

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

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

        18ml Solution A- 0.1M Citric Acid (Sigma, C1909) pH 2.5<\/span><\/p>

        82ml Reagent B- 0.1M Sodium Citrate (Sigma, S4641 ) pH 8.2<\/span><\/p>

        1L dH2<\/sub>O<\/span><\/p>

        pH 6.0<\/span><\/p>

        \u00a0<\/p>

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