{"id":17895,"date":"2018-01-25T13:47:31","date_gmt":"2018-01-25T13:47:31","guid":{"rendered":"https:\/\/research.cchmc.org\/lungimage\/?p=17895"},"modified":"2018-09-26T17:23:36","modified_gmt":"2018-09-26T17:23:36","slug":"3-year-old-old-human-lung-confocal-imaging-donor-d032","status":"publish","type":"post","link":"https:\/\/research.cchmc.org\/lungimage\/?p=17895","title":{"rendered":"3 Year-Old Old Human Lung Confocal Imaging SOX9, SOX2 and TUBB3"},"content":{"rendered":"\t\t
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3 Year-Old Old Human Lung Confocal Imaging SOX9, SOX2 and TUBB3<\/h1>\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t
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\"3<\/a>
3 Years-Old Human Lung Immunofluorescence and Confocal Imaging Donor D032 Showing Expressions of SOX9, SOX2, and TUBB3<\/div>
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3 Years-Old Human Lung Immunofluorescence and Confocal Imaging Donor D032 Showing Expressions of SOX9, SOX2, and TUBB3<\/div>
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3 Years-Old Human Lung Immunofluorescence and Confocal Imaging Donor D032 Showing Expressions of SOX9, SOX2, and TUBB3<\/div>
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3 Years-Old Human Lung Immunofluorescence and Confocal Imaging Donor D032 Showing Expressions of SOX9, SOX2, and TUBB3<\/div>
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3 Years-Old Human Lung Immunofluorescence and Confocal Imaging Donor D032 Showing Expressions of SOX9, SOX2, and TUBB3<\/div>
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3 Years-Old Human Lung Immunofluorescence and Confocal Imaging Donor D032 Showing Expressions of SOX9, SOX2, and TUBB3<\/div>
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3 Years-Old Human Lung Immunofluorescence and Confocal Imaging Donor D032 Showing Expressions of SOX9, SOX2, and TUBB3<\/div>
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3 Years-Old Human Lung Immunofluorescence and Confocal Imaging Donor D032 Showing Expressions of SOX9, SOX2, and TUBB3<\/div>
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3 Years-Old Human Lung Immunofluorescence and Confocal Imaging Donor D032 Showing Expressions of SOX9, SOX2, and TUBB3<\/div>
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3 Years-Old Human Lung Immunofluorescence and Confocal Imaging Donor D032 Showing Expressions of SOX9, SOX2, and TUBB3<\/div>
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3 Years-Old Human Lung Immunofluorescence and Confocal Imaging Donor D032 Showing Expressions of SOX9, SOX2, and TUBB3<\/div>
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3 Years-Old Human Lung Immunofluorescence and Confocal Imaging Donor D032 Showing Expressions of SOX9, SOX2, and TUBB3<\/div>
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3 Years-Old Human Lung Immunofluorescence and Confocal Imaging Donor D032 Showing Expressions of SOX9, SOX2, and TUBB3<\/div>
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3 Years-Old Human Lung Immunofluorescence and Confocal Imaging Donor D032 Showing Expressions of SOX9, SOX2, and TUBB3<\/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>

\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>SOX9<\/span><\/a>,\u00a0<\/span>SOX2<\/span><\/a>, and\u00a0<\/span>TUBB3<\/span><\/a><\/p>

Purpose:\u00a0\u00a0<\/span>Stain slides of 4\u00b5m frozen sections of human lung from HTC and from Pathology for\u00a0SOX9<\/span><\/a>,\u00a0<\/span>SOX2<\/span><\/a>, and\u00a0<\/span>TUBB3<\/span><\/a>\u00a0with antigen
retrieval.<\/p>

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

  1. Paraffin sections are placed at 60o<\/sup>C for 2 hours to overnight 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. 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 anti-SOX9<\/span><\/a>\u00a0(AB<\/span>\u20135535<\/span><\/a>, Lot # 2167153, Millipore) dilute 1:100, for mouse anti-SOX2<\/span><\/a>\u00a0(SC-365823<\/span><\/a>, Santa Cruz,
    Lot# C0317) dilute 1:100, for mouse anti-    TUBB3<\/span><\/a>\u00a0(anti-tubulin \u03b2 3<\/span><\/a>, cat. # 801201, Biolegend)\u00a0dilute 1:200 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, for anti-SOX9<\/span><\/a>) at 1:200, Goat
      Alexa Fluor 568 anti-mouse IgG1 (      A21124<\/span><\/a>, Lot# 1600878, for anti-SOX2<\/span><\/a>) at 1:200, Donkey Alexa Fluor 633 anti-mouse
      IgG2A\u00a0      A21136<\/span><\/a>, Lot# 1345055,\u00a0TUBB3<\/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. 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><\/ol>
      1. Store slides in light sealed box @ 4o<\/sup>C<\/li><\/ol>

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

        Tissue Used:<\/span>
        <\/span><\/span>LMH-16-D032-RLL-4A2.6
        <\/span>Gender: Female
        <\/span>Age: 3 years<\/span><\/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>

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

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