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Primary_Raphe_Cultures[1]

Primary_Raphe_Cultures[1] - Sunbola's Protocol Primary...

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Sunbola’s Protocol Primary Raphe Cultures Solutions needed:  Hanks Plating Media Hanks + FBS Matrigel Digerstion Solution  Trypsin Dissociation Solution 4/0-Ara C DNase PBS 1. Sterilize all tools in 70% ethanol a. Place a cover slip in wells and place 50ul of  Matrigel  in the center of each  {use a new tip/plate}  Incubate for >30 min 2. Sacrifice/Dissection: female rat at  E-15 [Materials: scissors, anesthetic,25 gauge  needle, ethanol, tissue, big tweezers, petri dish (add PBS to 1 big dish and Hanks  + FBS to a medium one)] a. Inject rat with .8 ml of nematol (~5 min) b. Cut rat open down and immediately remove embryos; place in Petri dish  c. Carefully remove embryos from sac and place in PBS dish d. Dissect out raphe and place in Hank+ FBS Petri dish{cut into tiny pieces  when complete} 3. Suck-up  Hank+ FBS+ raphe  with 10ml pipette and put in 15ml conical tube 4.
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