{"id":30520,"date":"2019-09-30T18:20:47","date_gmt":"2019-09-30T18:20:47","guid":{"rendered":"https:\/\/research.cchmc.org\/lungimage\/?p=30520"},"modified":"2019-09-30T18:29:06","modified_gmt":"2019-09-30T18:29:06","slug":"41-day-old-human-lung-immunofluorescence-and-confocal-imaging-donor-207-expressing-hopx-abca3-and-sftpc","status":"publish","type":"post","link":"https:\/\/research.cchmc.org\/lungimage\/?p=30520","title":{"rendered":"41 Day-Old Human Lung Immunofluorescence and Confocal Imaging Donor 207 Expressing HOPX, ABCA3, and SFTPC"},"content":{"rendered":"\t\t
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41 Day-Old Human Lung Immunofluorescence and Confocal Imaging Donor 207 Expressing HOPX, ABCA3, and SFTPC<\/h1>\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t
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\"41<\/a>
41 Day-Old Human Lung Immunofluorescence and Confocal Imaging Donor D207 Showing Expression of HOPX, SFTPC, and ABCA3<\/div>
20X<\/div><\/div>\"41<\/a>
41 Day-Old Human Lung Immunofluorescence and Confocal Imaging Donor D207 Showing Expression of HOPX, SFTPC, and ABCA3<\/div>
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41 Day-Old Human Lung Immunofluorescence and Confocal Imaging Donor D207 Showing Expression of HOPX, SFTPC, and ABCA3<\/div>
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41 Day-Old Human Lung Immunofluorescence and Confocal Imaging Donor D207 Showing Expression of HOPX, SFTPC, and ABCA3<\/div>
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41 Day-Old Human Lung Immunofluorescence and Confocal Imaging Donor D207 Showing Expression of HOPX, SFTPC, and ABCA3<\/div>
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41 Day-Old Human Lung Immunofluorescence and Confocal Imaging Donor D207 Showing Expression of HOPX, SFTPC, and ABCA3<\/div>
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41 Day-Old Human Lung Immunofluorescence and Confocal Imaging Donor D207 Showing Expression of HOPX, SFTPC, and ABCA3<\/div>
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41 Day-Old Human Lung Immunofluorescence and Confocal Imaging Donor D207 Showing Expression of HOPX, SFTPC, and ABCA3<\/div>
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41 Day-Old Human Lung Immunofluorescence and Confocal Imaging Donor D207 Showing Expression of HOPX, SFTPC, and ABCA3<\/div>
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41 Day-Old Human Lung Immunofluorescence and Confocal Imaging Donor D207 Showing Expression of HOPX, SFTPC, and ABCA3<\/div>
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41 Day-Old Human Lung Immunofluorescence and Confocal Imaging Donor D207 Showing Expression of HOPX, SFTPC, and ABCA3<\/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>

Immunofluorescence- HOPX, ABCA3, and SFTPC<\/strong><\/span><\/p>

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

Purpose:<\/strong>\u00a0\u00a0<\/strong>Stain slides of 5\u00b5m paraffin sections of human lung from the HTC for HOPX<\/b>, ABCA3, and SFTPC<\/strong>\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. Briefly equilibrate slides in Antigen Retrieval Buffer.<\/li>
  4. Antigen retrieval, pH 6.0 (times will vary according to microwave).<\/li>
  5. 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>
  6. Microwave according to instructions on microwave.<\/li>
  7. Cool on countertop, 15 min.<\/li>
  8. Rinse with dH2<\/sub>O<\/li>
  9. 1X PBS, 5 min.<\/li>
  10. Block in 4% Goat serum\/PBS-T, 2 hours at RT.<\/li>
  11. For Rabbit anti-HOPX <\/b>(SC-30216, Lot# C2212, Santa Cruz) dilute 1:100, for mouse anti-ABCA3 <\/b>(WMAB-17G524, Seven Hills Bioreagents) dilute 1:100, for guinea pig SFTPC<\/b>\u00a0(GP992, in house, bleed date 12.13.07) dilute 1:500 in blocking buffer. Spin down in\u00a0\u00b5fuge\u00a0for 20 minutes and incubate on tissue overnight @ 4 degrees 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# 188524,\u00a0 for anti-HOPX<\/b>), Goat Alexa Fluor 568 anti-mouse IgG2B (A21144<\/strong><\/a>, Lot# 1906480, for anti-ABCA3<\/strong>) at 1:200, Donkey Alexa Fluor 647 anti-guinea pig (706-605-148, for anti SFTPC<\/b>) 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. 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>
    10. Store slides in light-sealed box at 4o<\/sup>C.<\/li><\/ol>

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

      Tissue Used:
      <\/strong>LMH-D207-RLL-4A2.12<\/b>
      Gender: Female
      <\/strong>Age: 41 Days<\/strong><\/p>

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

      Appendix
      <\/strong>Antigen Retrieval Solution<\/strong><\/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>\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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      <\/div>\n\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\t<\/div>\n\t\t<\/div>\n\t\t\t\t\t<\/div>\n\t\t<\/section>\n\t\t\t\t<\/div>\n\t\t","protected":false},"excerpt":{"rendered":"

      Donor Tissue Kindly Provided by Dr. Gloria Pryhuber from\u00a0the University of Rochester Medical Center Immunofluorescence- HOPX, ABCA3, and SFTPC \u00a0 Purpose:\u00a0\u00a0Stain slides of 5\u00b5m paraffin sections of human lung from the HTC for HOPX, ABCA3, and SFTPC\u00a0with antigen retrieval. Day 1 Paraffin sections are placed at 60oC for 2 hours to overnight to melt paraffin. […]<\/p>\n","protected":false},"author":1,"featured_media":30489,"comment_status":"closed","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"footnotes":""},"categories":[1],"tags":[],"class_list":["post-30520","post","type-post","status-publish","format-standard","has-post-thumbnail","hentry","category-uncategorized"],"yoast_head":"\n41 Day-Old Human Lung Immunofluorescence and Confocal Imaging Donor 207 Expressing HOPX, ABCA3, and SFTPC -<\/title>\n<meta name=\"robots\" content=\"index, follow, max-snippet:-1, max-image-preview:large, max-video-preview:-1\" \/>\n<link rel=\"canonical\" href=\"https:\/\/research.cchmc.org\/lungimage\/?p=30520\" \/>\n<meta property=\"og:locale\" content=\"en_US\" \/>\n<meta property=\"og:type\" content=\"article\" \/>\n<meta property=\"og:title\" content=\"41 Day-Old Human Lung Immunofluorescence and Confocal Imaging Donor 207 Expressing HOPX, ABCA3, and SFTPC -\" \/>\n<meta property=\"og:description\" content=\"Donor Tissue Kindly Provided by Dr. Gloria Pryhuber from\u00a0the University of Rochester Medical Center Immunofluorescence- HOPX, ABCA3, and SFTPC \u00a0 Purpose:\u00a0\u00a0Stain slides of 5\u00b5m paraffin sections of human lung from the HTC for HOPX, ABCA3, and SFTPC\u00a0with antigen retrieval. Day 1 Paraffin sections are placed at 60oC for 2 hours to overnight to melt paraffin. 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