{"id":7306,"date":"2016-04-15T19:06:01","date_gmt":"2016-04-15T19:06:01","guid":{"rendered":"https:\/\/research.cchmc.org\/lungimage\/?p=7306"},"modified":"2018-10-02T18:55:59","modified_gmt":"2018-10-02T18:55:59","slug":"4-month-human-lung-grp-tubb3-and-muc5ac-confocal-imaging","status":"publish","type":"post","link":"https:\/\/research.cchmc.org\/lungimage\/?p=7306","title":{"rendered":"4 Month Human Lung GRP, TUBB3, and MUC5AC Confocal Imaging"},"content":{"rendered":"\t\t
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4 Month Human Lung GRP, TUBB3, and MUC5AC Confocal Imaging<\/h1>\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t
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\"4<\/a>
4 Month Old Human Lung Immunofluorescence and Confocal Imaging Showing Expression of Grp, TubB3, and Muc5ac Genes<\/div>
20x Tile Scan<\/div><\/div>\"4<\/a>
4 Month Old Human Lung Immunofluorescence and Confocal Imaging Showing Expression of Grp, TubB3, and Muc5ac Genes<\/div>
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4 Month Old Human Lung Immunofluorescence and Confocal Imaging Showing Expression of Grp, TubB3, and Muc5ac Genes<\/div>
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4 Month Old Human Lung Immunofluorescence and Confocal Imaging Showing Expression of Grp, TubB3, and Muc5ac Genes<\/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: GRP<\/span>, TUBB3<\/span>, and MUC5AC<\/span><\/span>\u00a0<\/span><\/p>

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

Purpose:\u00a0\u00a0<\/span>Stain slides of 5\u00b5m frozen sections of human lung from HTC and from Pathology for GRP<\/b>, TUBB3<\/span>, and MUC5AC<\/span>\u00a0with antigen retrieval.<\/p>

Day 1<\/strong><\/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% Goat serum\/PBS-T, 2 hours at RT.<\/li>
  10. For Rabbit anti-GRP<\/strong><\/a> (Immunostar<\/strong><\/a>-order from Fisher, Cat# 20073) dilute 1:100, for mouse anti-TUBB3<\/strong><\/a> (anti-tubulin \u03b2 3<\/strong><\/a>, cat. # 801201, Biolegend)\u00a0dilute 1:200, for mouse anti-MUC5AC<\/strong><\/a>\u00a0(Abcam, AB3649, Lot# GR135303-1) dilute 1:200 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-rabbit IgG (A11034<\/strong><\/a>, Lot# 1298480,\u00a0 for anti-GRP<\/strong><\/a>), Goat Alexa Fluor 568 anti-mouse IgG2A (A21134<\/strong><\/a> for anti-TUBB3<\/strong><\/a>) at 1:200, Goat Alexa Fluor 647 anti-mouse IgG1 (A21240<\/strong><\/a>, Lot# 1562685, anti-MUC5AC<\/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 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>

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

      Tissue Used:
      <\/strong>LMH-16-D024-RLL-6A2.16
      <\/strong>Gender: Male
      <\/strong>Age: 4 Months<\/strong><\/p>

      \u00a0<\/strong><\/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>

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