Wholemount in situ hybridisations on zebrafish embryos using digoxigenin probesAlex Schier 12/12/94
* Notes by Rolf Karlstrom and Atsushsi Kawakami in italics
* No DEPC treated water used, all embryo incubation buffers (also where not specifically mentioned) except paraformaldehyde contain 0.1% Tween 20
Probe synthesisThis is essentially according to the method recommended by Boehringer Mannheim. Cut 20 ug of plasmid DNA with appropriate enzyme in 100 ul. Pheno1, phenol/chloroform, chloroform and ethanol precipitate. Dissolve in 15 ul ddH20.
I recommend to add 200 ug/ml Proteinase K after enzyme digestion and incubate 30 min.
1. Mix together
2 ul linelized plasmid (1 ug of insert DNA);
2 ul 10 x transcription buffer
2 u1 10 x nucleotide mix
(10 mM each ATP, GTP, CTP, 6.5 mM UTP, 3.5 mM digoxigenin-UTP);
1 ul (20 units) of RNase inhibitor.
11 ul water
2 ul (40 units) T7,, T3 or SP6 RNA polymerase.Incubate the mixture for 2 hour at 37 oC.
10 x Transcription buffer
400 mM Tris-HCl pH 8.0;
60 mM MgC12;
100 mM dithiothreito1;
20mM spermidine;
100 mM NaCl;
RNase inhibitor 1 Unit/ul2. Take 2 ul for elecrophoresis. The probe can be checked on freshly made 1% mini-gel / TAE. The gel box should be washed before use. 2ul rxn mixture should contain 500 ng of RNA.
3. Add 2 ul (40 units) of DNase I and incubate at 37oC for 30 min. to remove the plasmld DNA.
Stop the reaction (optional) by adding 2 ul EDTA (0.2 M pH 8.0) and
Precipitate the RNA with 2.5 u1 4 M LiCl, and 75 ul pre-chilled ethanol for 30 min at ―80 oC.
4. Spin down the pellet and dissolve in 100 u1 of RNAse-free water. Add 900 ul of Hyb solution and store at-20 oC.
We have had success with probes of 0.5-2 kb and signals are stronger if they are not hydrolysed prior to use (requires proteinase K step!). I did not see any improvement by removing the nucleotides e.g. by NucTrap columns.
Preparation of embryos
Embryos are mixed during most incubation by pipetting up and down incubation media or by turning on wheel in Eppendorf tubes.
You can put up to 50 embryos in one tube.
Heavy mixing or media/air interface contact lead to damage of embryos.
1. Embryos younger than 20 somites are best left undechorionated and fixed o/n or longer in 4% paraformalde/PBS at 4 oC.
2. Wash twice 5 min in PBT and dechorionate manually.
3. After a few minutes of equilibration with 4-5 changes of methanol the embryos are stored at ―20 oC in Eppendorf tubes.
(Embryos can be stored in this way for several months or even years)
4. Rehyd,ate fixed embryos by soaking for 5 min each in
75% methanol + 25% PBT (1xPBS, 0.1% Tween 20).,
50% methanol + 50% PBT;
25% methanol + 75% PBT.
4x 5 min in 100% PBT
Young embryos (before lS) seem to be rather fragile during rehydration!!
Use some of these embryos to preabsorb the Anti-Dig Ab solution (put directly into 1:100 dilution
5. Incubate embryos in proteinase K at room temperature (10 ug/ml PBT.Stosk lmg/ml proteinase K in PBT at ―20 oC. Dilute l00 x before use).
5-10 min for pre lS,
10-15 min pre 16h,
15-20 min post 16h.
20-35 min post 36hr.
This step 6 should be titrated. Wash 2 times with PBT. >36h -- 20-30'
6. Refix embryos in 4% paraformaldehyde in lx PBS for 20 min. at room temperature.
7. Rinse 5x 5 min in PBT.
Hybridisation
1. Hybridisation buffer
50% formamide (up to 65% formamide to reduce background),
5xSSC (pH 7.0),
500 ut,or/ml torula (yeast) RNA,
50 ug/ml heparin,
0.1% Tween 20,
9mM citric acid to pH 6.0-6.52. Prehybridise embryos with ~500-1000 ul buffer for at least 1 hr (Preferably for 4-5 h) at hybridisation temperature (60-70oC). The hybridisation temperature is dependent on the probe but we have obtained good signals with all our probes at 65-70 oC in 50% formamide. Zebrafish krox-20 and pax-b also work very well at 70 oC in 65% formamide. Use heating block. or hybridisation oven or water bath for incubation and washing.
3. Replace prehybridisation buffer containing probe. We use 150 ng digoxigenin riboprobe in 200 ul hybridisation solution.
(preheated) as a starting point.
4. Incubate ovenight at 65-70 oC in a 1.5 ml microfuge tube Probes are stable in hyb and can be reused several times.
(Put in baskets for mass handling from here out)
5. Washes are all done at the hybridisation temperature with preheated solutions. Mix after 5 min by turning tube.
10 min 75% hyb + 25% 2x SSC;
10 min 50% hyb+ 50% 2x SSC;
10 min25% hyb + 75% 2x SSC;
10 min 100% 2xSSC
2x 30 min. 0.2x SSC at 70 oC.(down to 0.05 x SSC for higher stringency)
6. Perform the following washes at room temperature:5 min 75% 0.2x SSC + 25% PBT
5 min 50% 0..2x SSC + 50% PBT
5 min 25% 0.2x SSC + 75% PBT
5 min 100% PBT
Preabsorption of the anti-digoxigenin antibodies.(Skip this if used embyos to pre-absorb Ab)
The antibody must be preabsorbed against zebrafish embryos or adult zebrafish acetone powder
(Grind up several hundred day 2 embryos in minimal volume of egg water or freeze adult in liquid nitrogen and grind up. Add 4x vol cold acetone, leave on ice for 30 min. Spin down 10k 10 min. Discard S/N and wash again in acetone. Spin down, discard S/N and let embryo powder dry on filter paper. Pulverize and keep at 0.2% in 2 mg/ml BSA in PBT)
Add 1u1(0.75 units) antibody to 400 ul of 0.2% w/v zebrafish powder in 2 mg/ml BSA in PBT. Rotate sideways for at least 1 hour at room temperature, then spin in a microfuge before diluting to the desired concentration (1:2000-4000). The diluted antibody is stable under these conditions at 4 oC and we have reused it up to three times. Anti-fluorescin antibody coupled to HRP is used at a final 1:200 dilution of 0.15u/ul stock (i.e. twice as concentrated as anti-dig).
Antibody incubation and staining
7. Block with 2 mg/ml BSA, 5% sheep serum in PBT for a minimum of 60 min.
8. Incubate for 2 h at RT or o/n at 4 oC in 200 ul of a 1/2000 dilution (0.375 units/ml) of preabsorbed sheep anti-digoxygenin-alkaline phosphatase conjugated Fab fragments or 1/200 anti-fluororescin-HRP in 2mg/ml BSA PBT.
9. Wash for at least 2 h in 2mg/ml BSA PBT with 8 solution changes. (2 quick changes followed by 6 X 15 min washes)
10. Equilibrate 3x 5 min in freshly made NTMT buffer
(0.1 M T,is-HCl pH 9.5; 50 mM MgC12., 0.1 M NaCl; 0.1% Tween 20)
Stain with X-phosphate/NBT on shaker in dark (3.4 ul of 100 mg/ml NBT in 70% dimethylfomamide and 3.5 ul of 50 mg/ml X-phosphate in dimethylformamide added to 1 ml of NTMT buffer,). Take care that embryos do not stick together.
11. Stop reaction with washes in PBT. krox-20 comes up within minutes, hh and gsc take longer.
(can stop by post-fixing 1 hr in 4% PFA. This is important for a long lasting prep)
Clearing and embedding
(I mount directly in 70% glycerol for post-somite stage embryos).
1. Dehydrate 2x 5 min in methanol
2. Clear in benzylbenzoate/benzylalcohol = 2:1. Precipitate will slowly dissolve (within few days) in this solution. Keep in fridge. Mount in Permount and take pictures immediately after clearing. Embryos are very fragile.
Reagents
DIG RNA labelling mixture (10 x)
for 20 RNA samples Boehringer 1277 073
Anti-digoxygenin Fab fragment - akaline phosphatase
150 u Boehringer 1093 274
T3 RNA polymerase
1000 u + 10 x buffer Boehringer 1031 163
T7 RNA polymerase
1000 u + 10 x buffer Boehringer 881 767
RNase inhibitor
2000 u Boehringer 799 017
DNase I
RNase free 10000 u Boehringer 776 785
Proteinase K
100 mg Boehringer 745 723
1 x PBS
8 g NaCl
0.2 g KCl
1.44 g Na2HPO4
0.24 g KH2PO4 pH7.4 per liter
or 8 g NaCl
0.2 g KCl
3.63 g Na2HPO4 12H2O
2.4 g KH2PO4 pH 7.4 per liter
Make 10 x stock solution and autoclave1 x PBT
l x PBS + 0.1% Tween 20
4% paraformaldehyde / PBS
4 g in 100 ml PBS,
dissolve at 68 oC,
add a few drops of 5N NaOH, check pH 7.4.
Cool to RT and filter (0.45 um).
Make 10 ml aliquots and freeze at ―20 oC.20 x SSC
175.3 g NaCl;
88.2 g Natrium-citrate pH 7.0 per 1 LHyb
50% formamide Gibco BRL 500g 15515-026
5xSSC,
500 ug/ml torula (yeast) RNA Sigma 100g R-6625
50 ug/ml heparin Sigma H-7005
0.1% Tween 20
9mM citric acid to pH 6.0-6.5BSA
50g bovine albumin Sigma A-8022