{"content":[{"text":"One Color Section In­Situ Hybridization for Octopus bimaculoides","bbox":{"left":0.117564,"top":0.09026,"width":0.614922,"height":0.017070000000000002}},{"text":"Judit Pungor \n Host Lab: Dr. Clifton Ragsdale, University of Chicago \n Home Lab: Dr. Stuart Thompson, Hopkins Marine Station of Stanford University \n 5/1/12­7/31/12","bbox":{"left":0.117564,"top":0.129324,"width":0.636705,"height":0.073301}},{"text":"This protocol is a modification of one originally developed by the Ragsdale lab for in­situ \n hybridization (Rowell, 2010). While a number of changes have been made because of \n the challenges of octopus sections in­situ hybridization, protocol modification remains in \n progress. Further improvements will be published as they are established.","bbox":{"left":0.117564,"top":0.224821,"width":0.703113,"height":0.073301}},{"text":"The goal of this protocol is the detection of O. bimaculoides specific gene products \n within the neural tissue of the animal.","bbox":{"left":0.117564,"top":0.320318,"width":0.65898,"height":0.035102999999999995}},{"text":"­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­ \n ­­­­­­­­­­­­­","bbox":{"left":0.117564,"top":0.377617,"width":0.686818,"height":0.03510200000000002}},{"text":"Prepare sections \n 1. Cut PFA fixed, sucrose­sunk tissue on a freezing microtome (25­50 µm). Dry \n sections well (2 hrs. or overnight at RT).","bbox":{"left":0.117564,"top":0.431095,"width":0.642893,"height":0.05706699999999998}},{"text":"Day One: (RNase­free) \n 1. Place slides in RNase­free mailers. \n 2. Fix in 4% fresh paraformaldehyde (PFA) in PBS for 15 min. \n 3. Wash in DEPC­PBS at RT for 5 min. (X3) \n 4. Incubate in Proteinase K (1µg/ml in Proteinase K Buffer) at 37°C for 30 min. \n 5. Fix in 4% PFA in PBS for 15 min. \n 6. Wash in DEPC­PBS for 5 min. (X3) \n 7. Wash in TEA 10 min. (X1) \n 8. Add 125 uL Acetic Anhydride to 50uL of TEA. (Always add Acetic Anhydride to \n TEA fresh before each wash.) Wash slides in Acetic Anhydride in TEA 10 min. (X3) \n 9. Wash DEPC­PBS 5 min. (X1) \n 10. Hybridization: Add approximately 15 ml of prehybridization solution to each \n mailer. Slides may be stored at ­20°C at this point or incubate slides, following warming \n to room temperature if stored at ­20°C, in prehybridization solution in a 72°C water bath \n for at least 60 minutes. \n 11. Empty the warmed prehybridization solution into a fresh Falcon tube, add 100 µl \n of probe, mix well, and add to the appropriate slide mailer. \n 12. Incubate at 72°C overnight.","bbox":{"left":0.117564,"top":0.510358,"width":0.697136,"height":0.34069499999999997}},{"text":"Day Two","bbox":{"left":0.122515,"top":0.869429,"width":0.07569,"height":0.01600299999999999}}],"images_bbox":[],"images_bbox_not_text_overlap":[]}