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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