Whole-mount in situ Hybridization for zebrafish gonads B. Draper ...

Whole-mount in situ Hybridization for zebrafish gonadsB. DraperUpdated: 7Dec2006I. Preparation of DIG/FITC-labeled RNA Probe.A. DNA Template Preparation(1) Linearize 5-10 ug plasmid with enzyme (avoid enzymes that leave 3’ overhang)(2) phenol/chloroform extract(3) EtOH ppt.(4) Resuspend in ddH 2 O at 500ng/ulB. RNA Probe Synthesis11ul2ul2ul2ul1ul0.5ul1ul20ulddH 2 O to 20ul10X Transcription Buffer10X DIG or FITC rNTP mix500ng/ul linearized DNA template (1ug total)100mM DTTRNasinRNA PolymeraseV t(1) 37 o C 1hrs(2) Add 1µl RNA polymerase, incubate additional 1 hr.(3) Add 0.8ul 0.5M EDTA(4) Remove 1 ul to run on gel.(5) Add 30 ul DEPC-ddH2O->Purify RNA over a G50-sephadex spin column.(6) Add 5ul 5M LiCl(7) Add 188 ul EtOH(8) Incubate –20ºC for 30 min.(9) Pellet in microfuge and wash pellet 1X in 70% EtOH(10) Resuspend in 100ul DEPC-ddH2O (or 80 ul if hydrolyzing: see below)(11) Store at –80ºC.C. Probe Hydrolysis (Optional)Note: for probes longer than 1.5-2.0 kb, signal to noise can be improved by hydrolyzingprobe to a smaller size.(1) Resuspend pellet from above in 80 ul DEPC-H2O(2) Add:10ul 0.4M Na Bicarbonate (1.68 gm/50 ml)10ul 0.6M Na Carbonate (3.72 gm monohydrate/ 50 ml)-> Incubate at 60ºC for t min.where t min= (start kb-desired kb)/0.11(start kb)(desired kb)Note: A good desired size is 0.3-0.4 kb(3) Add:400 ul DEPC-H2O34 ul 3M NaOAc2.6 ul Glacial HOAc1.1 ml EtOH (-20ºC)(4) Incubate at –20ºC for 30min.(5) Resuspend pellet in 100 ul DEPC-H2O and store at -80º(6) Analyze 5ul on gel to access cleavage

II. in situ HybridizationA. Fixation and Preparation of gonads(1) Fix gonads in situ in 1X Fix (4% paraformaldehyde in PBS) overnight at 4 o C.-Prepare adults by decapitating (cutting just behind the pectoral fins), and cutting along theventral midline to open the coelomic cavity. To preserves optimum morphology, removegonads from fish only after they have been fixed.-For fish

Solutions:PBSTw50 ml 10X PBS2.5 ml 20% Tween 20ddH 2 O to 500 mlPre-hybridization Solution[Final]25 ml Formamide 50%12.5 20X SSC 5X50 ul 50 mg/ml Heparin 50 ug/ml500 ul 50 mg/ml tRNA 500 ug/ml250 ul 20% Tween 20 0.1%460 ul 1M Citric acid ~pH 6.0ddH 2 O to 50 mlColoration Buffer[Final]5 ml 1M Tris-HCl, pH 9.5 100mM2.5 ml 1M MgCl 2 50mM1 ml 5M NaCl 100mM250 µl 20% Tween 20 0.1%ddH 2 O to 50 ml