Protocol for high-resolution whole mount in situ hybridization using 3’-DIG labeled miRCURY™ probes
Method prepared by Dr. Erno Wienholds and Dr. Wigard Kloosterman, the Plasterk Group, Hubrecht Laboratory, Utrecht, The Netherlands
A. Fixation and storage of embryos: Remove chorions by pronase treatment (for embryos older than 18 somites) or manually (for earlier stages). We only do manual dechorionation on all stages. Fix embryos in 4% paraformaldehyde (PFA) in PBS overnight at 4°C. Transfer embryos into 100% Methanol (MeOH), store them at -20°C (2h to several months).
B. In situ hybridization, day 1:
Rehydration: Transfer embryos into small baskets and rehydrate by successive incubations in:
75% MeOH - 25% PBS for 5 min
50% MeOH - 50% PBS for 5 min
25% MeOH - 75% PBS for 5 min
100% PBST (PBS/Tween-20 0.1%) 4 x 5 min Digest with Proteinase K (10 μg/ml).
2.1. blastula and gastrula: 30 seconds
2.2. early somitogenesis: 1 min
2.3. late somitogenesis (14 to 22 somites): 5 min
2.4. 24h embryos: 15 min
2.5. 36h/48h embryos: 30 min Refix in 4% PFA-PBS, 20 min. Wash in PBST, 5 x 5 min. Preadsorb the anti-DIG antibody (Boehringer) in a 1:1000 dilution in PBST-sheep serum 2%-BSA (2mg/ml) for several hours at RT with a batch of previously fixed embryos. Use about 500 embryos for 10 ml of antibody. Prepare the Prehybridization and Hybridization mix:
C. Prehybridization and Hybridization mix (HM):
Formamide 50-65%
5 x SSC
Tween-20 0.1%
Citric acid to pH 6.0 (460 μl of 1M stock for 50 ml)
Heparin 50 μg/ml
tRNA 500 μg/ml
Note: Add tRNA and Heparin to the prehybridation and hybridization mix only (not the wash solutions). Vary the formamide concentration according to the desired hybridization stringency.
Prehybridize embryos in 800 μl of hybridization mix, 2 to 5 hrs at a temperature which is ca. 20-22°C below the calculated melting temperature (Tm) of the miRCURY™ probe. Label 100 pmol of miRCURY™ probe (LNA probe) for miRNA detection using the DIG Oligonucleotide 3’-End Labeling Kit from Roche Applied Science (cat # 3 353 575) according to the manufacturer’s instructions with the following modifications: 8.1. Use 200 U of terminal transferase (0.5 μl) for each end-labeling reaction and incubate the reaction mixture for 30 min at 37 °C. Place on ice and stop the reaction by adding 5 μl of 0.1 M EDTA (pH 8.0). 8.2. Remove the unincorporated label from the 3’-DIG labeled miRCURY™ probe in a volume of 25 μl using a MicroSpin G-25 column (Amersham Biosciences cat# 27-5325-01) according to the manufacturer’s instructions. Note: It is important to clean-up the labeled probe before use, since the unincorporated label may result in unspecific background staining in situ hybridization. Remove prehybridization mix, discard, and replace with 200 μl of hybridization mix containing 1-2 μl of the MicroSpin G-25-purified 3’-DIG labeled miRCURY™ probe. Adjust the temperature of the waterbath so that the in situ hybridization is carried out at a temperature which is ca. 20-22°C below the calculated melting temperature (Tm) of the miRCURY™ probe and hybridize overnight.
D. In situ hybridization, day 2: Washes: 100% HM at the same temperature as above for hybridization (C.10, adjusted according to the Tm of the miRCURY™ probe), very brief wash 75% HM/25% 2 x SSC at the same temperature as in D.1, 15 min 50% HM/50% 2 x SSC at the same temperature as in D.1, 15 min 25% HM/75% 2 x SSC at the same temperature as in D.1, 15 min 2 x SSC at the same temperature as in D.1, 15 min 0.2 x SSC, at the same temperature as in D.1, 2 x 30 min 75% 0.2 (or 0.05) x SSC/25% PBST at RT, 10 min 50% 0.2 (or 0.05) x SSC/50% PBST at RT, 10 min 25% 0.2 (or 0.05) x SSC/75% PBST at RT, 10 min PBST at RT, 10 min PBST/2% sheep serum/2mg:ml BSA at RT, several hrs
E. Incubation with anti-DIG antiserum: Incubate in antibody solution overnight with agitation at +4°C.
F. Anti-DIG antibody solution:
Pre-adsorbed anti-DIG, 1:5000 dilution (final concentration) in PBST
2% sheep serum 2mg/ml BSA G.I. Zebrafish in situ hybridization, day 3: Washes: Remove antiserum, discard, and then wash extensively: PBST at RT, very brief wash PBST at RT, 6 x 15 min Staining buffer (100 mM Tris HCl pH9.5, 50 mM MgCl2, 100 mM NaCl, 0.1% Tween 20), at RT 3 x 5 min
Staining: Incubate embryos in staining solution at RT and monitor with a dissecting microscope.
G.II. Mouse in situ hybridization, day 3: Washes: Remove antiserum, discard, and then wash extensively: PBST at RT, very brief wash PBST at RT, 5 x 1 hour PBST at +4 °C, 2 days by exchanging the PBST buffer at every 2 hours Staining buffer (100 mM Tris HCl pH9.5, 50 mM MgCl2, 100 mM NaCl, 0.1% Tween 20), 3 x 5 min
Staining: Incubate embryos in staining solution at RT and monitor with a dissecting microscope.
Staining solution: NBT 50 mg/ml - 225 μl BCIP 50 mg/ml - 175 μl Staining buffer - 50 ml (NBT stock: 50 mg Nitro Blue Tetrazolium in 0.7 ml of Dimethyl-formamide anhydride + 0.3 ml H2O. BCIP stock: 50 mg of 5-Bromo 4-Chloro-3-Indolyl Phosphate in 1ml anhydrous Dimethyl-formamide). 2. Stop the reaction by removing the staining solution and washing the embryos in: Incubate in antibody solution overnight with agitation at +4°C. Pre-adsorbed anti-DIG, 1:5000 dilution (final concentration) in PBST Stop solution: PBS pH5.5 EDTA 1mM 3. Store the embryos in stop solution at +4°C in the dark. Mounting: For observation using a dissecting microscope, mount embryos directly in stop solution and methylcellulose. For observation using a compound microscope, mount embryos in 100% glycerol. For embryos at early development stage (up to 18h), dehydrate in 100% methanol, clear for a few minutes in methylsaly-cilate, and mount in Permount. (What we mostly do) Wash embryos 3x 5 min in PBST. Dehydrate by successive incubations in:
75% PBS - 25% MeOH for 5 min
50% PBS - 50% MeOH for 5 min
25% PBS - 75% MeOH for 5 min
100% MeOH for 5 min
100% MeOH for 5 min
Murray’s (benzylalcohol:benzylbenzoate 1:2) for 5 min
Murray’s (benzylalcohol:benzylbenzoate 1:2), store at 4oC
Reagents and chemicals: PFA: paraformaldehyde (Sigma) 10 x PBS Tween-20 (Sigma P1379) PBST: PBS containing 0.1 % Tween-20 MeOH: methanol Proteinase K (Boehringer 1000144) Anti-DIG antibody - alkaline phosphatase Fab fragment (Boehringer 1 093 274) BSA fraction V protease free (Sigma A-3294) Formamide (deionized, high purity grade) 20 x SSC Heparin at 5 mg/ml (Sigma H3393) RNAse free tRNA (Sigma R7876, 50 mg/ml resuspended in H2O and extensively extracted several times in Phenol/CHCl3 to remove protein) Citric acid 1M Normal Sheep serum (Jackson ImmunResearch 013-000-121) Tris HCl pH9.5 1M MgCl 2 1M NaCl 5M NBT 50 mg/ml (made from powder, Sigma N6876) BCIP 50 mg/ml (made from powder, Sigma B8503) PBS pH5.5 EDTA 0.5M Glycerol 100% Methylsalicylate (Sigma M6752) Permount (Fisher SP15-100) This protocol is adapted from: Thisse, C., Thisse, B., Schilling, T. F., and Postlethwait, J. H. (1993). Structure of the zebrafish snail1 gene and its expression in wild-type, spadetail and no tail mutant embryos. Development 119, 1203-1215
Please refer to:
Thisse B, Heyer V, Lux A, Alunni V, Degrave A, Seiliez I, Kirchner J, Parkhill JP, Thisse C. Spatial and temporal expression of the zebrafish genome by large-scale in situ hybridization screening. Methods Cell Biol. 2004;77:505-19. |
|
