{"id":20020,"date":"2018-03-15T18:56:36","date_gmt":"2018-03-15T18:56:36","guid":{"rendered":"https:\/\/research.cchmc.org\/lungimage\/?p=20020"},"modified":"2018-11-12T16:05:47","modified_gmt":"2018-11-12T16:05:47","slug":"8-month-old-human-lung-confocal-imaging-donor-d086-fn1-foxf1-and-acta2","status":"publish","type":"post","link":"https:\/\/research.cchmc.org\/lungimage\/?p=20020","title":{"rendered":"8 Month-Old Human Lung Confocal Imaging Donor D086 FN1, FOXF1, and ACTA2"},"content":{"rendered":"\t\t
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8 Month-Old Human Lung Confocal Imaging Donor D086 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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\"8<\/a>
8 Month-Old Human Lung Immunofluorescence and Confocal Imaging Donor D086 Showing Expressions of FN1, FOXF1, and ACTA2<\/div>
20X<\/div><\/div>\"8<\/a>
8 Month-Old Human Lung Immunofluorescence and Confocal Imaging Donor D086 Showing Expressions of FN1, FOXF1, and ACTA2<\/div>
60X<\/div><\/div>\"8<\/a>
8 Month-Old Human Lung Immunofluorescence and Confocal Imaging Donor D086 Showing Expressions of FN1, FOXF1, and ACTA2<\/div>
60X<\/div><\/div>\"8<\/a>
8 Month-Old Human Lung Immunofluorescence and Confocal Imaging Donor D086 Showing Expressions of FN1, FOXF1, and ACTA2<\/div>
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8 Month-Old Human Lung Immunofluorescence and Confocal Imaging Donor D086 Showing Expressions of FN1, FOXF1, and ACTA2<\/div>
60X<\/div><\/div>\"8<\/a>
8 Month-Old Human Lung Immunofluorescence and Confocal Imaging Donor D086 Showing Expressions of FN1, FOXF1, and ACTA2<\/div>
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8 Month-Old Human Lung Immunofluorescence and Confocal Imaging Donor D086 Showing Expressions of FN1, FOXF1, and ACTA2<\/div>
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8 Month-Old Human Lung Immunofluorescence and Confocal Imaging Donor D086 Showing Expressions of FN1, FOXF1, and ACTA2<\/div>
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8 Month-Old Human Lung Immunofluorescence and Confocal Imaging Donor D086 Showing Expressions of FN1, FOXF1, and ACTA2<\/div>
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8 Month-Old Human Lung Immunofluorescence and Confocal Imaging Donor D086 Showing Expressions of FN1, FOXF1, and ACTA2<\/div>
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8 Month-Old Human Lung Immunofluorescence and Confocal Imaging Donor D086 Showing Expressions of FN1, FOXF1, and ACTA2<\/div>
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8 Month-Old Human Lung Immunofluorescence and Confocal Imaging Donor D086 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 Immunofluorescence-\u00a0<\/span>FN1<\/span><\/a>,\u00a0<\/span>FOXF1<\/span><\/a>, and\u00a0<\/span>ACTA2<\/span><\/a><\/span><\/p>

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

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

Day 1<\/span><\/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-<\/span>FN1<\/span><\/a>\u00a0(AB2413<\/span><\/a>, ABCAM, anti-Fibronectin<\/span><\/a>) dilute 1:100, For goat anti-\u00a0FOXF1<\/span><\/a>\u00a0dilute 1:100, for mouse anti-ACTA2<\/span><\/a>\u00a0
    (    \u03b1 Smooth muscle actin<\/span><\/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<\/span><\/p>

    1. Rinse slides in PBS-T 3X, 5 min.<\/li>
    2. Apply secondary antibody, Donkey Alexa Fluor 488 anti-rabbit IgG (A21206<\/span><\/a>, Lot# 1608521, anti-FN1<\/span><\/a>)<\/span>\u00a0Donkey Alexa Fluor 568
      anti-goat IgG (      A11057<\/span><\/a>, Lot# 1485187, for anti-FOXF1<\/span><\/a>), at 1:200, Donkey Alexa Fluor 647 anti-mouse IgG (A31571<\/span><\/a>, Lot#
      1549801,\u00a0      ACTA2<\/span>)<\/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.<\/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.<\/li><\/ol>
      1. Store slides in light sealed box @ 4o<\/sup>C<\/li><\/ol>

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

        Tissue Used:
        <\/span>LMH-15-D086-LLL-8B2.6
        <\/span>Age: 8 Months
        <\/span>Gender: Male<\/span><\/p>

        \u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\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>

        pH 6.0<\/p><\/div>

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