Whole mount in situ hybridization with digoxygenin probes
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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
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