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2. Material and Methods
Abbreviations: hr, hour(s); min, minute(s); RT, room temperature; WT, wild-type. Obtaining Xenopus laevis embryos:
1. Inducing ovulation: ovulation is induced by injection of 800 units of human chorionic gonadotropin (hCG) into the dorsal lymph sac of female frogs. After 12-16hr, eggs are collected by squeezing the animals with a firm and gentle touch. Eggs are collected in a Petri dish containing 0.1xMMR (10xMMR: NaCl 0.1M, KCl 2mM, MgSO4 1mM, CaCl 2mM, HEPES 5mM, pH 7.8 ), and should be fertilized immediately.
2. Isolating testes: a male frog is anaesthetized with 0.05% MS222 (3-aminobenzoic acid ethyl ester methanesulphonate salt, Sigma) in distilled water at RT for 20min. The animal is then washed and placed ventral side up in a box with ice. A small incision is cut with scissors in abdomen flanking, yellow fat bodies are pull out and the testes is isolated. The testes is then stored in 1xMMR with Gentamycin (50μg/mL, Sigma) at 4°C.
3. In vitro fertilization: just prior to fertilization, remove most of the solution with a pipette. Use a scissor to cut a small piece of testes, mix with eggs immediately. Let stand for 5min, then fill with some drops of 0.1xMMR to allow eggs to rotate.
4. Dejellying embryos: for microinjection, the jelly coat should be removed after fertilization. Dejelly solution: Tris-HCl 0.2M, pH8.8 with addition of DTT (3μM). Let stand at RT for 5min until the jelly coat is visible in the solution and embryos start to pack, rinse embryos with 0.1xMMR gently and thoroughly. Keep in 0.1xMMR for growth.
Capped-mRNA preparation and microinjection:
1. In vitro transcription of capped-mRNA using the mMESSAGE mMACHINE® kit is performed according to manufacturer’s instructions (Ambion).
2. During embryo development, prepare injection materials (mRNA), adjust injector and needle.
3. Select good embryos at desired stage into Petri dish with 4% Ficoll in 0.1xMMR (Ficoll can prevent leakage). Move embryos with hair loop gently, nudge and rotate embryos into the wells of plastic grid attached to the dish until they are all aligned and ready to be injected. 4. After injection, transfer the injected embryos in a clean dish with 4% Ficoll/0.1xMMR to
allow embryos to grow until stage 8-9, then replace solution with 0.1xMMR and leave embryos to grow to the desired stage for collection or fixation.
Animal cap assay:
Allows to investigate the effects of factors of interest on the determination of cell fates in the pluripotent animal cap embryonic stem (ACES) cells.
1. Microinjection: mRNAs are injected into the animal pole of both blastomeres of two-cell stage embryos, and cultivated in 4% Ficoll/0.1xMMR at 14°C.
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2. When embryos reach blastula stages (stage 8/9), remove the vitelline membranes from the embryos by using a pair of sharp forceps and dissect animal caps by using Gastromaster instrument (XENOTEK Engineering). All manipulations are carried out in 1xMBS solution (10xMBS: NaCl 0.88M, KCl 0.01M, MgSO4 8mM, HEPES 0.1M, NaHCO3 0.024M) with
addition of CaCl2 (0.7mM) and Gentamycin (50μg/mL), and all Petri dishes are precoated
with 1% agarose in sterile water.
3. Assemble three pieces of explants together on agarose-coated dishes to form conjugates that facilitate healing and healthy growing. Transfer explants to a fresh 1xMBS solution with addition of CaCl2 and Gentamycin to avoid contamination.
4. Always collect some WT embryos from the same batch and culture in parallel with the injected embryos and explants, in order to stage the explants and use as host embryos for transplantation assay.
5. In vitro cultivate the explants either to stage 15 or later stages such as stage 39, by reference to sibling wild-type embryos, followed by in vivo transplantation assay or molecular analyses, respectively.
In vivo transplantation assay of ACES cells:
Allows to test the eye-forming potential of treated ACES cells in host WT embryos
1. When explants of ACES cells and sibling WT embryos reach stage 15, remove the vitelline membrane with forceps then, using Gastromaster, remove from the WT embryos the eye field tissue on one side or a small piece of the posterior neural tube.
2. Explants at the same stage are cut in half with Gastromaster. One piece is inserted into of the cavity obtained by the surgery on the WT embryo, making sure it contacts the lateral wall. All manipulations are carried out in 1xMBS solution with addition of CaCl2 and Gentamycin,
and all dishes are coated with 1% agrarose in sterile water.
3. After overnight culture in 1xMBS, the completely healed transplants are transfered to fresh 0.1xMMR with Gentamycin, and cultured to stage 42. Embryos are then fixed and processed by in situ hybridization, immunohistochemistry or H-E staining.
Whole-mount in situ hybridization:
1. Fix the embryos at desired stage in MEMFA (MOPS 0.1M, EGTA 2mM, MgSO4 1mM,
3.7% formaldehyde) for 1hr at RT on the rotator
2. Remove MEMFA, dehydrate gradually into 100% ethanol, then store at -20°C. Day1:
3. Rehydrate embryos gradually by 5min washes in: 75% ethanol 25% H2O
50% ethanol 50% H2O
25% ethanol 75% 1xPBSTw (1xPBS, 0.1% Tween-20) 100% 1xPBSTw
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4. Wash 2x5min in 1xPBSTw with slow horizontal agitation
5. Permeabilize the embryos with proteinase K (stock is 10mg/ml, final concentration is 10μg/mL) for 10min at RT with slow horizontal agitation. Take care that the embryos are well dispersed, and keep to the precise incubation time.
6. Wash twice briefly with 1xPBSTw, rotating the vial in the hand
7. Refix the embryos with 4% paraformaldehyde in 1xPBS, rotate carefully the vial in the hand to mix well and incubate for 20min at RT with occasional hand agitation.
8. Wash twice briefly with1xPBSTw, then 4x5min with slow agitation.
9. Replace 1xPBSTw with Hybridization Mix, leave for a few minutes then replace with 100% Hybridization Mix.
10. Prehybridize for 2-4hr at 62°C.
11. Denature the DIG-labeled probe in 10μL MQ-water for 2 minutes at 90°C, add Hybridization Mix (preheated to 62°C) to make the probe at the final concentration of 0.1μg/mL.
12. Replace Hybridization Mix with 0.5mL Hybridization mix with probe, hybridize overnight at 62°C.
Day2:
13. Replace probe with recycled Hybridization Mix, 62°C for10min 14. Wash with 2xSSC /0.1% CHAPS for 2x30min at 62°C.
15. Wash with 0.2xSSC /0.1% CHAPS for 2x30min at 62°C.
16. Replace washing buffer with 1mL/vial of 50% 0.2xSSC/0.1% CHAPS-50% 1xMABT, leave for a few minutes.
17. Wash twice in 1xMABT (1xMAB, 0.1% Tween-20) buffer for 10min each at RT.
18. Antibody blocking: incubate the embryos in 0.5mL of 1xMABT + 2% Blocking Reagent + 10% heat-inactivated lamb serum + Egg extract for 3hr at RT, at the same time, prepare the antibody solution: anti-Digoxygenin coupled with alkaline phosphatase diluted at 1:2500-1:4000 in 1xMABT + 2% Blocking Reagent + 10% heat-inactivated lamb serum+ Egg extract, preincubate for 2hr at 4°C
19. Replace blocking buffer with antibody solution, and incubate overnight at 4°C on the rotator.
Day3:
20. Discard antibody solution and wash the embryos for 30 seconds in 1xMABT, then for at least 5x1hr in 1xMABT with agitation at RT. An overnight wash at 4°C is preferable.
Day4:
21. For the chromogenic reaction with alkaline phosphatase, wash twice 10min each at RT with alkaline phosphatase buffer:
100mM Tris, pH9.5 50mM MgCl2
100mM NaCl 0.1% Tween 20
2mM Levamisol (an inhibitor of endogenous phosphatase, add just before use)
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develop staining at RT.
23. When staining is adequate, replace the BM Purple with 1xPBSTw for 10min, then fix in MEMFA for 1hr at RT.
24. Dehydrate the embryos into 100% ethanol.
25. Bleaching embryos: replace the 100% ethanol with 70% ethanol for 5min, then replace in 50% ethanol + 50% 1xSSC for 5min. Bleach the embryos in bleaching solution: 2% H2O2,
0.5xSSC and 5% formamide. Put the glass vial under an incandescent lamp, cover with foil. In about 1hr, pigmented embryos turn white.
26. Transfer the embryos back to 100% ethanol and photograph.
In situ hybridization on cryostat sections:
1. Fix the tissue or embryos at desired stage in 4% paraformaldehyde in 1xPBS for 20-30min at RT on the rotator.
2. Remove the fixative, cryoprotect with 20% sucrose in 1xPBS, then embed the tissue into OCT.
3. Cut the tissue into sections at the thickness of 12μm, air dry and store at -80°C.
Hybridization:
4. Defrost sections at RT (at least half an hour). 5. Wash in 1xPBS for 5min at RT to remove the OCT.
6. Dilute DIG-labeled probe in hybridization buffer (prewarmed to 65°C) to a final concentration of 0.1-1μg/mL (I usually use 1μg/mL, and decrease the concentration if it gives a high background). Denature probe mix for 5-10min at 70°C.
7. Add 150ul of probe mix to each slide and cover the slide with a coverslip.
8. Hybridize overnight at 65°C in a sealed perspex box with 2 sheets of Whatman paper wetted with 1xSalts/ 50% formamide (10xSalts: 114g/L NaCl, 14.04g/L Tris-HCl, pH7.5, 1.34g/L Tris-base, 7.8g/L NaH2HPO4·2H2O, 7.1g/L Na2HPO4, EDTA 50mM, pH8).
Post-hybridization washes:
9. Transfer slides into a slide rack and put the rack in a box containing enough washing solution to allow the coverslips to fall off the slides.
Washing solution: 1xSSC, 50% formamide, 0.1% Tween-20, prewarmed to 65°C. 10. First wash for 15min at 65°C to allow coverslipes to fall off.
11. Wash 3x30min at 65°C.
12. Rinse in 1xMABT for 2x30min at RT.
Blocking and antibody staining:
13. Dry the slides off around the sections.
14. Block in 1xMABT + 2% Blocking Reagent + 20% heat-inactivated lamb serum, for at least 1hr at RT without a coverslip.
15. Antibody staining: 1/2500 dilution of anti-Digoxygenin in 1xMABT + 2% Blocking Reagent + 20% heat-inactivated lamb serum. Add 150μL on each slide and cover with a coverslip. Incubate in a humidified chamber at RT overnight.
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16. Wash sections at RT in 1xMABT, 5x30min.
17. Rinse sections in alkaline phosphatase buffer minus NBT/BCIP, 2x10min rocking at RT.
Staining reaction:
18. Transfer slides in a humidified chamber or equivalent, and add 180μl of NBT/BCIP staining buffer per slide, cover with Parafilm cut into the appropriate size. Check staining under the microscope at any moment.
Staining buffer: 18μL of NBT/BCIP stock solution (Roche) in 1mL alkaline phosphatase buffer
19. Stop the staining reaction by rinsing with 1xPBS 20. Mount in Aqua-Poly/Mount (Polysciences).
Immunohistochemistry on cryostat sections:
1. Fix the tissue or embryos at desired stage in 4% paraformaldehyde in 1xPBS for 20-30 min at RT on the rotator.
2. Remove the fixative, cryoprotect with 20% sucrose in 1xPBS, then embed the tissue into OCT.
3. Cut the tissue into sections at the thickness of 12μm, air dry and store at -80°C. 4. Defrost sections at room temp (at least half an hour).
5. Wash in 1xPBS for 5min.
6. (Optional) block with 1xPBS, 0.5%BSA, 0.1% Triton for 1hr at RT.
7. Incubate with primary antibody in 1xPBS, 0.5%BSA, 0.1%Triton for 1hr at 37°C (or 2hr at RT).
8. Wash 3x5 minutes in 1xPBS, 0.1%Triton.
9. Incubate with secondary antibody for 45min at 37°C (or 1.5hr at RT). 10. Wash 2x10min in 1xPBS, 0.1%Triton.
11. Incubate with nuclear staining dye (Hoechst: 1:1000 in 1xPBS, 0.1% Triton), for 5min at RT. 12. Wash for 10min in 1xPBS
13. Mount in Aqua-Poly/Mount.
Hematoxylin - Eosin staining on paraffin sections:
1. Embed embryos in paraffin and cut into sections at the thickness of 14μm. 2. Dewax the sections with Xylene, twice 10min each.
3. Rehydrate the sections gradually: 100% ethanol, 2x5min
90% ethanol, 5min 75% ethanol, 5min 50% ethanol, 5min
4. Wash slides in 1xPBS for 5min. 5. Wash in ddH20 for 1min.
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6. Stain with Hematoxylin solution for 5min (Sigma Accustain Harris Hematoxylin, Catalog HHS-32).
7. Rinse with running tap water for 5min.
8. Rinse with Acid alcohol (0.5% HCl in 70% ethanol, pH1.48) for 30 seconds. 9. Rinse with running tap water for 1min.
10. Treat with Blueing agent (0.1% Sodium Bicarbonate) for 2min. 11. Rinse with running tap water for 30 seconds.
12. Stain with 5% Eosin solution for 15min (Sigma Accustain Eosin Y, Catalog HT110-2-32). 13. Dehydrate the sections in ethanol gradually:
70% ethanol for 1min 90% ethanol for 1min 95% ethanol for 1min 100% ethanol for 2min
14. Wash with Xylene, twice 1min each. 15. Mount in Eukitt.
EdU incorporation and detection:
EdU (5-ethynyl-2’-deoxyuridine) is a nucleoside analog of thymidine and is incorporated into DNA during active DNA synthesis, allowing the detection of proliferating cells.
(Click-iT™ EdU Imaging Kits, Invitrogen)
1. When the sibling embryos reach stage 35, the explants are partially cut with Gastromater in 1xMBS solution with addition of EdU at the final concentration of 30μM, then left to grow to stage 39 (more than 10 hours).
2. Fix the animal caps in 4% paraformaldehyde in 1xPBS for 20min at RT.
3. Remove the fixative, cryoprotect with 20% sucrose in 1xPBS, then embed into OCT. 4. Cut the block into sections at the thickness of 12μm, air dry and store at -80°C. 5. Defrost sections at room temp (at least half an hour).
6. Wash in 1xPBS for 5min.
7. Wash twice with 3% BSA in 1xPBS, 10min each at RT. 8. Permeablize with 1xPBS, 0.3% Triton for 20min at RT. 9. Wash once with 3% BSA in 1xPBS, 10min.
10. Incubate with Reaction Cocktails in dark for 30min at RT. 11. Wash once with 3% BSA in 1xPBS, 10min.
12. Mount in Aqua-Poly/Mount.
Light-response analyses:
1. For c-fos expression analysis: both the 20pg-noggin eye field transplants and WT embryos were grown on a 12hr light / 12hr dark cycle (LD12:12) starting immediately after transplantation, and they were fixed either after the light on for 1 hour or after the light off
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for 1 hour, respectively, then analyzed for c-fos expression at stage 43 by in situ hybridization.
2. For electrophysiology: embryos were dark-adapted overnight, and eyes dissected under dim
red light. Retinae from 20pg-noggin eye field transplants and WT embryos were finely chopped and stored for 90min in darkness in Ringer solution (NaCl 113 mM, KCl 2.5 mM, CaCl2 1 mM, MgCl2 1.6 mM, EDTA 0.01 mM, glucose 10 mM, HEPES 3 mM, pH 7.7, with
30 U/mL DNase (Sigma-Aldrich) and 200 nM 9-cis-retinal (Sigma-Aldrich) to promote rhodopsin generation from free opsin. Retinal fragments were then transferred to the recording chamber and responses to light acquired using the suction pipette technique.
RT-PCR analysis:
Injected ACES cells with or without treatment were harvested at the indicated stages and frozen. Total RNAs were extracted by NucleoSpin RNAII (MACHEREY-NAGEL) and reverse transcription was performed by using Superscript III Reverse Transcriptase (Invitrogen) in the presence of poly-T primers. PCR was then performed using GoTaq DNA Polymerase (Promega). Cycling conditions were: 95°C, 2min; then 95°C, 30 seconds; 58°C, 30 seconds; 72°C, 30 seconds, for 30 cycles and ended with a single extension step of 72°C for 8min.
