{"id":5961,"date":"2016-03-14T19:31:09","date_gmt":"2016-03-14T19:31:09","guid":{"rendered":"https:\/\/research.cchmc.org\/lungimage\/?p=5961"},"modified":"2018-08-17T23:13:42","modified_gmt":"2018-08-17T23:13:42","slug":"pnd01-c57bl6-calca-scgb1a1-and-tuba1a-confocal-imaging","status":"publish","type":"post","link":"https:\/\/research.cchmc.org\/lungimage\/?p=5961","title":{"rendered":"Postnatal Day 1 C57BL6 CALCA, SCGB1A1, and TUBA1A Confocal Imaging"},"content":{"rendered":"

\"Post<\/a>
Post Natal Day 1 Confocal Imaging Showing Protein Expression of Calca, Scgb1a1, and Tuba1a Genes<\/div>
20x Tile Scan<\/div><\/div>\"Post<\/a>
Post Natal Day 1 Confocal Imaging Showing Protein Expression of Calca, Scgb1a1, and Tuba1a Genes<\/div>
20x<\/div><\/div>\"Post<\/a>
Post Natal Day 1 Confocal Imaging Showing Protein Expression of Calca, Scgb1a1, and Tuba1a Genes<\/div>
60x<\/div><\/div>\"Post<\/a>
Post Natal Day 1 Confocal Imaging Showing Protein Expression of Calca, Scgb1a1, and Tuba1a Genes<\/div>
20x<\/div><\/div>\"Post<\/a>
Post Natal Day 1 Confocal Imaging Showing Protein Expression of Calca, Scgb1a1, and Tuba1a Genes<\/div>
60x<\/div><\/div>\"Post<\/a>
Post Natal Day 1 Confocal Imaging Showing Protein Expression of Calca, Scgb1a1, and Tuba1a Genes<\/div>
20x<\/div><\/div>\"Post<\/a>
Post Natal Day 1 Confocal Imaging Showing Protein Expression of Calca, Scgb1a1, and Tuba1a Genes<\/div>
60x<\/div><\/div><\/div><\/div><\/b><\/p>\n<\/div>
<\/div><\/div><\/div><\/div><\/div>
\n\n\n\n\n\n
Gene
\n<\/strong><\/span><\/td>\n		<\/td>\n<\/td>\n<\/td>\n<\/td>\n<\/td>\n<\/tr>\n
Calca<\/a><\/strong><\/span><\/td>\nLungGens<\/strong><\/a><\/span><\/td>\n Ensemble<\/a><\/strong><\/span><\/td>\nGenecards<\/a><\/strong><\/span><\/td>\nNCBI<\/a><\/strong><\/span><\/td>\nThe Human Protein Atlas<\/a><\/strong><\/span><\/td>\n<\/tr>\n
Scgb1a1a <\/strong><\/span><\/td>\nLungGens<\/strong><\/a><\/span><\/td>\nEnsemble<\/a> <\/strong>
\n<\/strong><\/span><\/td>\n		Genecards<\/a><\/strong><\/span><\/td>\nNCBI<\/a><\/strong><\/span><\/td>\nThe Human Protein Atlas<\/a><\/strong><\/span><\/td>\n<\/tr>\n
Tuba1a<\/strong><\/a><\/span><\/td>\nLungGens<\/strong><\/a><\/span><\/td>\nEnsemble<\/strong><\/a><\/span><\/td>\nGenecards<\/strong><\/a> <\/span><\/td>\n NCBI<\/strong><\/a> <\/span><\/td>\nThe Human Protein Atlas<\/a><\/strong><\/span><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n

Day 1<\/strong><\/p>\n

    \n
  1. For Frozen tissue, rinse 2X in PBS, then 5 min in 4% PFA\/PBS, then rinse 1X in PBS. Briefly equilibrate slides in Antigen Retrieval Buffer.<\/li>\n<\/ol>\n
      \n
    1. 10 mM sodium citrate, pH 6.0, and heat in a microwave for 15 min at 90o<\/sup>C.\u00a0*We usually do a series of three runs to equal the time\/temp because of evaporation (refill coplin jars with dH2<\/sub>O).<\/li>\n
    2. Microwave according to instructions on microwave.<\/li>\n
    3. Cool on countertop, 15 min.<\/li>\n
    4. Rinse with dH2<\/sub>O<\/li>\n
    5. 1X PBS, 5 min.<\/li>\n
    6. Block in 4% Goat serum\/PBS-T, 2 hours at RT.<\/li>\n
    7. For mouse anti \u2013CALCA<\/strong><\/a> (C7113, Sigma<\/strong>)\u00a0dilute 1:100 in blocking buffer, For guinea pig anti-SCGB1A1<\/strong> (G210, Bl. 3\/24\/11) dilute 1:1500, for mouse anti-TUBA1A<\/strong> <\/a>(T7451, Sigma)\u00a0dilute 1:1000 in blocking buffer. Spin for 10 minutes in a \u00b5fuge, apply to tissue, and incubate on tissue overnight @ 4o<\/sup>C.<\/li>\n<\/ol>\n

       <\/p>\n

      Day 2 <\/strong><\/p>\n

        \n
      1. Rinse slides in PBS-T 3X, 5 min.<\/li>\n
      2. Apply secondary antibody , Goat Alexa Fluor 488 anti-mouse IgG1 (A21121<\/strong><\/a>, Lot# 1345050, for anti-CALCA<\/strong><\/a> ) dilute 1:200, Goat Alexa Fluor 555 anti-guinea pig IgG (A21435<\/strong><\/a>, Lot# 1571699, CCSP) at 1:200, Donkey Alexa Fluor 647 anti-mouse IgG2B (<\/strong>A21242<\/strong><\/a>, Lot# 1148959, anti-TUBA1A1<\/strong><\/a>) at 1:200, in blocking buffer. Vortex and \u00b5fuge at full speed for 10 minutes. Incubated at room temperature for 1 hour.<\/li>\n
      3. Rinse in PBS-T 3X, 5 min.<\/li>\n
      4. Dilute DAPI 1:2000 and apply to slides for 10 min.<\/li>\n
      5. Wash in PBS-T 3X, 5 min.<\/li>\n
      6. Rinse slides in 0.1M PB or TB, 3X, 5 min.<\/li>\n
      7. Add 1 drop of Prolong Gold anti-fade mounting medium (P36930).<\/li>\n
      8. Coverslip with Gold Seal Coverslip (Cat# 3422 Electron Microscopy Sciences, 22 X 22 mm).<\/li>\n
      9. Allow Prolong Gold to cure overnight at room temp in light sealed box.<\/li>\n
      10. Store slides in light sealed box at 4o<\/sup><\/li>\n<\/ol>\n

         <\/p>\n

         <\/p>\n

         <\/p>\n

        Tissue Used: <\/strong>\u00a0\u00a0 LMM.15.66A.6.73<\/strong><\/p>\n

        PND01 C57BL6<\/strong><\/p>\n

        Gender: Female<\/strong><\/p>\n

        Weight= 1.14g<\/strong><\/p>\n<\/div>

        <\/div><\/div><\/div><\/div><\/div><\/p>\n","protected":false},"excerpt":{"rendered":"","protected":false},"author":1,"featured_media":26063,"comment_status":"closed","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"footnotes":""},"categories":[274,106],"tags":[43,11,46,44,28,121,45],"class_list":["post-5961","post","type-post","status-publish","format-standard","has-post-thumbnail","hentry","category-c57bl6","category-p01-confocal","tag-calca","tag-ccsp","tag-cilia","tag-neuroendocrine","tag-scgb1a1","tag-sma","tag-tuba1a"],"yoast_head":"\nPostnatal Day 1 C57BL6 CALCA, SCGB1A1, and TUBA1A Confocal Imaging -<\/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=5961\" \/>\n<meta property=\"og:locale\" content=\"en_US\" \/>\n<meta property=\"og:type\" content=\"article\" \/>\n<meta property=\"og:title\" 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