Whole mount in situ hybridization with digoxygenin probes

Whole mount in situ hybridization with digoxygenin probes

1. Probe labelling (DNA) DNA template (100 ng - 1 µg ) in dH20 5 µl pd(N)6 (10 mg/ml) 10 µl Mix template and hexamer, boil 5 min, chill on ice 3 min then add : 10 X Hexanucleotide buffer (or Vial 5 of Genius kit) 2 µl dig-dNTP mix ( Vial 6 of Genius kit ) 2 µl Klenow 1 µl Incubate at 37 C o/n Add 1 µl Klenow and incubate for an additional 4 hr. Stop reaction with : 0.5 M EDTA 2 µl Yeast tRNA (1 mg/ml ) 20 µl 4 M LiCl 4 µl EtOH 120 µl Precipitate on dry ice 30' Spin 10' in cold Wash in 70% EtOH 2X, Dry in speed vac Resuspend in 100 µl hybridization solution and store at -20 C until use. 2. Preparation of samples A. Embryos : Harvest eggs into a nested screen, rinse with dH2O + 0.02% Triton X-100. Dechorionate with 50% bleach (2') , rinse again. Transfer into a 15ml capped tube. B. Ovaries : Dissect ovaries in EBR. Transfer whole ovaries into a 15 ml capped tube and rinse with EBR. 3. Fixation Add to the 15 ml tube : Fixing solution (Fixing sol.= 0.1 M Hepes, pH 6.9, 2 mM MgSO4, 1mM EGTA) 1.6 ml 20% Paraformaldehyde 0.4 ml Heptane 8 ml Shake vigorously for 20' Remove Fixing solution and add 10 ml MeOH (the embryos or ovaries should sink to the bottom). Samples may be stored in this way at 4 C for up to 3 weeks. 4. Rehydration Transfer embryos with some MeOH into Eppendorf tubes. Wash with ME (MeOH : EGTA : 90% MeOH ; 10% 0.5 M EGTA pH 8). Rehydrate the samples as follows : 5' in 700 µl ME + 300 µl PP ( 4% Paraformaldehyde in PBS, dilute from 20% stock-20 C) 5' in 500 µl ME + 500 µl PP 5' in 300 µl ME + 700 µl PP 20' in 1 ml PP Wash samples 3X in PBT ( PBS + 0.1% Tween 20), 5' each 5. Proteinase treatment Dissect whole ovaries into ovarioles Incubate samples in 50 µg/ml Proteinase K in PBT at RT for 8-10'. Wash 1X for 5' with 2 mg/ml Glycine in PBT and 2X for 5' with PBT Refix with PP for 20'. Wash 3X for 5' with PBT. 6. Hybridization Add 100 µg/ml denatured salmon sperm DNA and 50 µg/ml heparin to hybridization solution before use. Prewash the samples as follows : 5' with 200 µl Hyb sol : PBT = 1:1 5' with 100 µl Hyb sol Prehybridize with 100 µl Hyb sol at 45 - 50 C > 1 hr. Denature probe by boiling 5'; chill on ice 3' Hybridize with probe in 50-100 µl Hyb. sol at 45 - 50 C O/N. 7. Washes SAVE THE PROBE!!! All washes done at 50 C with preheated solutions Rinse with 500 µl Hyb. sol Wash with 500 µl Hyb. sol 20' Wash with 500 µl Hyb sol:PBT = 1:1 20' Wash 4X with PBT 5' / wash 8. Detection A. Antibody staining Dilute Ab 1:10 in PBT, preadsorb with fixed ovaries or embryos > 1 hr, dilute 1:200 with PBT before use Incubate samples in Ab sol for 1 hr at RT or overnight at 4 C Wash 2X 5' with PBT Wash 2X 5' with AP buffer B. Color reaction To 1ml AP buffer add : 4.5 µl NBT (vial 9 of Genius kit) 3.5 µl X- Phosphate (vial 10 of Genius kit) Incubate samples in color reaction solution for the desired time, about 45 min. Rinse 4 x 5' with PBT and mount in 50% Glycerol BUFFERS 1. Hexamer priming mix (pd(N)6, Pharmacia ; 50 A260 units) 50 A260 U / 21.7 A 260/ mg = 2.304 mg. Dissolve this in 230 µl DEPC-ddH2O to give 10 mg/ml. Divide into 50 ul aliquots and freeze at -20 C. 2. Hexanucleotide Buffer (or vial 5 of Genius kit) 0.5 M Tris-HCl (pH 7.2) 500 µl (1 M Tris) 1 mM DTT 5 µl (0.2 M) 0.1 M MgCl2 100 µl (1 M) 2 mg/ml BSA 40 µl (50 mg/ml) 3.1 mg /ml Hexanucleotide 310 µl ( 10 mg/ml) DEPC-ddH2O 45 µl 1 ml 3. 10X DIG-dNTP Mix ( vial 6 of Genius kit) 1 mM dATP 1.0 µl (100 mM) 1 mM dCTP 1.0 µl (100 mM) 1 mM dGTP 1.0 µl (100 mM) 0.65 mM dTTP 0.65 µl (100 mM) 0.35 mM dig-dUTP 3.5 µl (10 mM) DEPC-H2O 92.85 µl 100.0 µl 4. HYB (store at -20 C) HYB WASH STOCKS 50% deionized formamide 5.0ml 5.0 ml 100%, -20 C 5X SSC 2.5 ml 2.5 ml 20X SSC, RNase free 100 µg/ml autoclaved DNA 100 µl - 10 mg/ml 100 µg/ml tRNA 100 µl - 10 mg/ml, -20 C 50 µg/ml heparin 10 µl 10 µl 50 mg/ml, -20 C 0.1% Tween 20 100 µl 100 µl 10% DEPC-ddH2O 2.2 ml 2.4 ml 10 ml 10 ml 5. Heparin Na Heparin (Sigma H-3125). Dissolve in DEPC-ddH2O at 50µg/ml. Store in 100µl aliquots at -20 C. 6. PBT 50 ml DEPC-10X PBS 5 ml DEPC-ddH2O 45 ml Autoclave 10% Tween 20 0.5 ml (0.1% final) 7. PBT plus glycine 5 ml DEPC-10X PBS 0.5 ml 10 mg/ml glycine 1 ml (2 mg/ml final) 10% Tween 20 50 µl (0.1% final) DEPC-H2O 3.5 ml 8. Paraformaldehyde - 20% Stock 20% (w/v) paraformaldehyde (solid stored at 4 C) in PBS. Incubate at 65 C until dissolved. Store at -20 C. 9. AP Buffer 100 ml final 1 M Tris, pH 9.5 10 ml 0.1 M 1 M MgCl2 5 ml 0.05 M 5 M NaCl 2 ml 0.1 M H2O 83 ml 10. 10X PBS (one liter) NaCl 200.0 g KCl 5.0 g KH2PO4 5.0 g Na2HPO4-2 H2O 27.8 g

上一篇：Crude Extract Immunodepletion   下一篇：Antibody Staining of Ovaries