{"content":[{"text":"1. Transfer slides to Day 2 mailers containing pre­heated Solution X. For the first \n Solution X Wash, add 5mg/mL of Yeast tRNA to the Solution X before adding it to the \n mailers. Mailers with hybridization solution and probe can be saved at ­20°C and \n reused. \n 2. Stringent Wash: Solution X for 45 min. at 72°C. (X3) \n 3. Wash for 15 min. in TBST at RT. (X3) \n 4. While washing in TBST, prepare the preadsorbed antibody: Incubate 3­5 mg of \n octopus embryo powder in 0.5ml of TBST for 30 minutes at 72°C. Chill on ice for 15 \n minutes. Add 5µl of lamb serum and 3 µl anti­DIG alkaline phosphatase fab fragments. \n Incubate at 4°C for 60 minutes while rocking. Centrifuge for 10 minutes and remove \n supernatant containing preadsorbed antibody. \n 5. Block for 1 hr. at RT (or overnight at 4°C): 10% lamb serum in TBST. \n 6. Add preadsorbed Anti­DIG antibody to 15 ml of 1% lamb serum in TBST. Mix \n well and incubate at RT for 2 hrs. (or overnight at 4°C). \n 7. 15 min. TBST wash. (X3) \n 8. Wash for 10 min. in freshly prepared NTMT. \n 9. Color Reaction: Add 3.5 µl NBT and 3.5 µl BCIP per ml NTMT. Incubate at RT in \n the dark. Leave overnight in color reaction at RT or 4°C as appropriate. (To remove \n pink or light purple background, replace color reaction solution with plain NTMT and \n leave at 4°C overnight.)","bbox":{"left":0.117564,"top":0.089215,"width":0.703335,"height":0.378893}},{"text":"Day Three: \n 1. Replace color reaction solution with fresh solution once a day or as appropriate \n to minimize background. When color has developed, rinse twice in Stop TE buffer, then \n wash in Stop TE for 1­2 hrs. \n 2. Wash in TBST overnight at RT. \n 3. Store in 10% Formalin in PBS overnight at 4°C. \n 4. Wash in PBS for 5 min. at RT. (X2) \n 5. Remove slides from mailers, place in slide holders and allow them to dry at RT.","bbox":{"left":0.117564,"top":0.490304,"width":0.7008610000000001,"height":0.14969899999999997}},{"text":"Solutions","bbox":{"left":0.117564,"top":0.657515,"width":0.09021299999999999,"height":0.01707000000000003}},{"text":"Hybridization Solution (500mL) \n 50% Formamide: 250 ml Formamide \n 5x SSC: 12.5 ml 20x SSC (pH 4.5) \n 1% SDS: 50 ml 10% SDS \n .2 mg/mL heparin: .1 g \n .5 mg/mL Acetylated BSA: 8.3 mL of ~30mg/mL stock \n 5 mg/mL Yeast tRNA: 2.5 g","bbox":{"left":0.117564,"top":0.691804,"width":0.43003899999999995,"height":0.13346400000000003}},{"text":".1M TEA pH 8 (for acetylation) (1L) \n 13.3 mL triethanolamine \n 983.7mL DEPC­H2O","bbox":{"left":0.117564,"top":0.842689,"width":0.295762,"height":0.05706699999999998}}],"images_bbox":[],"images_bbox_not_text_overlap":[]}