{"id":574,"date":"2015-02-03T03:59:58","date_gmt":"2015-02-03T03:59:58","guid":{"rendered":"http:\/\/joekitz.com\/LungMap\/?p=574"},"modified":"2016-11-18T14:15:29","modified_gmt":"2016-11-18T14:15:29","slug":"cchmc-mouse-soptimed-matings-and-tissue-harvestfixationprocessing","status":"publish","type":"post","link":"https:\/\/research.cchmc.org\/lungimage\/?p=574","title":{"rendered":"CCHMC Mouse SOP Timed Matings and Tissue Harvest, Fixation, and Processing"},"content":{"rendered":"

CCHMC Mouse SOP:\u00a0 Timed Matings & Tissue Harvest\/Fixation\/Processing<\/strong><\/p>\n

A.\u00a0 MICE<\/strong><\/p>\n

1.\u00a0 C57BL\/6J are obtained or derived from the Jackson Laboratory colony.<\/p>\n

2.\u00a0 Breeding females should be between 8-15 weeks of age and no older than 6 \u00a0months.<\/p>\n

3.\u00a0 Male mice can be used up to 7-8 months of age, or until no longer producing pregnancies.<\/p>\n

4.\u00a0 Expect seasonal changes in breeding performance. Some strains produce more litters in the spring and summer than in winter and fall.<\/p>\n

B.\u00a0\u00a0 BREEDING<\/strong><\/p>\n

1.\u00a0\u00a0\u00a0\u00a0 Females should have one litter before setting up breedings for experimental material.\u00a0 NOTE:\u00a0 First litters may not be representative of the average number of pups per litter for the strain.<\/p>\n

2.\u00a0\u00a0\u00a0\u00a0 Place 1 to 3 females into each male\u2019s cage in the late afternoon, prior to the dark-cycle. Record date and time.<\/p>\n

3.\u00a0\u00a0\u00a0\u00a0 Check for vaginal plugs early the next morning (plug will dissolve over time).<\/p>\n

4.\u00a0\u00a0\u00a0\u00a0 By convention, the date a plug is observed is designated as gestational day 0 or 0.5.\u00a0\u00a0 We use 0.5 (or E0.5).\u00a0 Record date and time.<\/p>\n

5.\u00a0\u00a0\u00a0\u00a0 Remove male from cage.<\/p>\n

6.\u00a0\u00a0\u00a0\u00a0 Separate unplugged female mice from the male.\u00a0\u00a0 Do not use these female mice for breeding until it can be demonstrated that there is no pregnancy.\u00a0 This can be done by monitoring daily and\/or by palpitation.<\/p>\n

C.\u00a0\u00a0 HARVESTING<\/strong><\/p>\n

1.\u00a0\u00a0\u00a0\u00a0 Harvest embryos in the morning (before noon) on the desired gestational day.<\/p>\n

2.\u00a0\u00a0\u00a0\u00a0 Obtain fetuses by hysterectomy.\u00a0 Dam should be anesthetized and then exsanguinated to prevent congestion of blood in the tissues.<\/p>\n

3.\u00a0\u00a0\u00a0\u00a0 Remove pups from uterus and maintain in a large weigh boat on ice.\u00a0 As the pups are extracted from the uterus, place each pup in a smaller weigh boat on ice, and number or label for future identification.<\/p>\n

4.\u00a0\u00a0\u00a0\u00a0 Strip way extra-fetal membranes.<\/p>\n

5.\u00a0\u00a0\u00a0\u00a0 Weigh and measure crown-to-rump (C-R) length for each pup prior to fixation and record. NOTE: Variations in lung development within a litter are directly correlated with fetal body weight and crown-to-rump length measurements.<\/p>\n

6.\u00a0\u00a0\u00a0\u00a0 Compare body weights and C-R lengths with fetal growth tables to determine developmental age.\u00a0 See attached or below.<\/p>\n

7.\u00a0\u00a0\u00a0\u00a0 Remove a piece of tail or tissue for isolating genomic DNA.<\/p>\n

D.\u00a0 FIXATION AND PROCESSING<\/strong><\/p>\n

1.\u00a0\u00a0\u00a0\u00a0 For E12.5 to E14.5, leave embryos intact and immerse in 20 ml of 4% PFA in 1XPBS (formulated with DEPC-treated water) in a 50 ml sterile tissue culture tube.\u00a0 Fix at room temperature for one hour, and then at 4oC overnight. Number\/label tubes accordingly.\u00a0 NOTE: Volume of fixative should be at least 10-20 times the volume of the tissue.\u00a0 Container should be at least 2-3 times the diameter of the tissue.\u00a0 Fixative should cover fetus<\/p>\n

2.\u00a0\u00a0\u00a0\u00a0 Larger pups between E16 and postnatal day 5 should be kept on ice as above, weighed and measured, and then exsanguinated.<\/p>\n

3. \u00a0 \u00a0 Open the abdomen and clip the descending aorta and\/or vena cava to let the animal bleed out.<\/p>\n

4. \u00a0 \u00a0 Divide the pup into two segments with a sharp razor blade at the level of the diaphragm, cutting just below the inferior extent of the thorax or below the liver.<\/p>\n

\u00b7\u00a0\u00a0\u00a0\u00a0\u00a0 Remove liver and diaphragm, and trim rib cage.\u00a0 NOTE:\u00a0 You must strip off the diaphragm prior to immersion in the fixative in order to allow the fixative to enter the chest cavity directly.<\/p>\n

\u00b7\u00a0\u00a0\u00a0\u00a0\u00a0 If leaving the chest intact, the head may be removed by bisecting it between the upper and lower jaws.\u00a0 The lower jaw will still be attached to the neck.\u00a0 This prevents the larynx and trachea from retracting.<\/p>\n

\u00b7\u00a0\u00a0\u00a0\u00a0\u00a0 Alternately, remove lung from the chest.<\/p>\n

\u00b7\u00a0\u00a0\u00a0\u00a0\u00a0 Place in 20 ml of 4% PFA\/1X PBS, incubate at room temperature for 1 hour, and then incubate at 4oC overnight.\u00a0 Number\/label containers accordingly.\u00a0 NOTE: If the fixative becomes bloody from the initial dissection, then rinse and replace with fresh fixative.<\/p>\n

3.\u00a0\u00a0\u00a0\u00a0 Wash 3 times in 1X PBS (formulated with DEPC-treated water) for 10 minutes each at room temperature.<\/p>\n

4.\u00a0\u00a0\u00a0\u00a0 Incubate in 30% Sucrose in 1X PBS (formulated with DEPC-treated water) at 4oC.<\/p>\n

5.\u00a0\u00a0\u00a0\u00a0 The day before freezing place in 2 parts 30% Sucrose\/1XPBS and 1 part OCT, and incubate at 4oC.<\/p>\n

6.\u00a0\u00a0\u00a0\u00a0 Freeze in molds filled with OCT on dry ice or in isopentane that has been pre-chilled in a container surrounded by dry ice.<\/p>\n

7.\u00a0\u00a0\u00a0\u00a0 Alternately, embryos can be prepared for paraffin embedding.\u00a0 Following step 3, the tissue is dehydrated through 30% and 50% ETOH for 10 minutes each, and then 70% ETOH, 3 times, 10 minutes each.\u00a0 All solutions are prepared with DEPC-treated water.\u00a0 Tissue can then be stored in 70% ETOH at 4oC until processed for paraffin embedding.<\/p>\n

8.\u00a0\u00a0\u00a0\u00a0 Embed each fetus so that it is resting on its back with the heart facing up.\u00a0 When embedding E18.5, lift up the fetus so the fetus is resting more on the shoulder blade.<\/p>\n

E.\u00a0 RNA EXTRACTION<\/strong><\/p>\n

1.\u00a0\u00a0\u00a0\u00a0 Alternatively, tissue can be harvested for RNA extraction. Extract the lungs from the chest and place in 1X PBS.<\/p>\n

2.\u00a0\u00a0\u00a0\u00a0 Carefully, dissect each lobe away from the heart, trachea, bronchi, esophagus.<\/p>\n

3.\u00a0\u00a0\u00a0\u00a0 Place in collection tube and immediately place on dry ice. Store at -80oC until RNA can be isolated.<\/p>\n

F.\u00a0 SOLUTIONS FOR FIXATION \/ PROCESSING<\/p>\n

1.\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 PBS (phosphate-buffered saline)<\/p>\n

10X stock solution, 1 liter, pH 7.2 to 7.4*\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 20X stock solution<\/p>\n

80\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 g\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 NaCL\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 160 g \u00a0\u00a0\u00a0 NaCl<\/p>\n

2\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 g\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 KCl\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 \u00a0\u00a0\u00a0\u00a0 4 g \u00a0\u00a0 KCL<\/p>\n

11.5\u00a0\u00a0\u00a0 g\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 Na2HPO4-7H2O\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 \u00a0\u00a0 23 g \u00a0\u00a0 Na2HPO4-7H2O<\/p>\n

2\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 g\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 KH2PO4\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 \u00a0\u00a0\u00a0\u00a0 4 g \u00a0\u00a0 KH2PO4<\/p>\n

2.\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 4% paraformaldehyde in PBS<\/p>\n

Must be made up fresh, i.e. on the same day as fixation.<\/p>\n

(a)\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 Recipe #1 – for small volumes.<\/p>\n

10\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 ml of 16% paraformaldehyde stock solution, EM grade*<\/p>\n

4\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 ml of 10X PBS (prepared as above)<\/p>\n

26\u00a0\u00a0\u00a0\u00a0\u00a0 ml of D2H2O<\/p>\n

40\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 ml total (pH 7.2-7.4)<\/p>\n

(b)\u00a0\u00a0\u00a0\u00a0\u00a0 Recipe #2 – for larger volumes.<\/p>\n

Add 8 g to 100 ml of water (ddH2<\/sub>O).\u00a0 Heat to 60\u00b0C in a fume hood.\u00a0 Add a few drops of 1 N NaOH to help dissolve.\u00a0 When the solid has completely dissolved, let the solution cool to room temperature, and add 100 ml of 2X PBS and adjust pH to 7.4.\u00a0 This solution should be prepared fresh.\u00a0 You may have to filter solution if a precipitate persists.\u00a0 We have also frozen batches of this solution at -20\u00b0C for subsequent use.<\/p>\n

These are basic recipes.\u00a0 You will have to make more if fixing a large number of animals.\u00a0 Figure 20 ml per fetus (or 10X the volume of the specimen).\u00a0 You may fix the tissue in sterile 50 ml tissue culture tubes.<\/p>\n

*obtain from Electron Microscopy Sciences, #15710,10 X 10 ml per box, 1-800-523-5874.<\/p>\n

G.\u00a0\u00a0 Gender Identification PCR<\/strong><\/p>\n

http:\/\/www.karger.com\/Article\/FullText\/348677<\/a><\/p>\n

McFarlane L, Truong V, Palmer J, S, Wilhelm D, Novel PCR Assay for Determining the Genetic Sex of Mice. Sex Dev 2013;7:207-211<\/p>\n

SXF\u00a0 5\u02b9-GATGATTTGAGTGGAAATGTGAGGTA-3\u02b9<\/p>\n

SXR, 5\u02b9-CTTATGTTTATAGGCATGCACCATGTA-3\u02b9<\/p>\n

PCR Conditions<\/p>\n

94o<\/sup>C\u00a0\u00a0\u00a0\u00a0 2 minutes<\/p>\n

94o<\/sub><\/sup>C<\/p>\n

57o<\/sup>C \u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 \u00a0\u00a0\u00a030 Seconds, 35 cycles<\/p>\n

72o<\/sup>C<\/p>\n

72o<\/sup>C\u00a0\u00a0 5 minutes<\/p>\n

4o<\/sup>C\u00a0\u00a0\u00a0\u00a0 Hold<\/p>\n","protected":false},"excerpt":{"rendered":"

CCHMC Mouse SOP:\u00a0 Timed Matings & Tissue Harvest\/Fixation\/Processing A.\u00a0 MICE 1.\u00a0 C57BL\/6J are obtained or derived from the Jackson Laboratory colony. 2.\u00a0 Breeding females should be between 8-15 weeks of age and no older than 6 \u00a0months. 3.\u00a0 Male mice can be used up to 7-8 months of age, or until no longer producing pregnancies. […]<\/p>\n","protected":false},"author":1,"featured_media":0,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"footnotes":""},"categories":[4],"tags":[8,18,27,33,34,35],"class_list":["post-574","post","type-post","status-publish","format-standard","hentry","category-sop","tag-breeding","tag-gender-pcr","tag-rna-extraction","tag-tissue-fixation","tag-tissue-harvest","tag-tissue-processing"],"yoast_head":"\nCCHMC Mouse SOP Timed Matings and Tissue Harvest, Fixation, and Processing -<\/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=574\" \/>\n<meta property=\"og:locale\" content=\"en_US\" \/>\n<meta property=\"og:type\" content=\"article\" \/>\n<meta property=\"og:title\" content=\"CCHMC Mouse SOP Timed Matings and Tissue Harvest, Fixation, and Processing -\" \/>\n<meta property=\"og:description\" content=\"CCHMC Mouse SOP:\u00a0 Timed Matings & Tissue Harvest\/Fixation\/Processing A.\u00a0 MICE 1.\u00a0 C57BL\/6J are obtained or derived from the Jackson Laboratory colony. 2.\u00a0 Breeding females should be between 8-15 weeks of age and no older than 6 \u00a0months. 3.\u00a0 Male mice can be used up to 7-8 months of age, or until no longer producing pregnancies. 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