New developed fluorescent 2 color Whole mount in situ hybridization produces a clear and high S/N ratio image to analyze mRNA localization inside 1 cell and in tissue levels.
Method Article
Fluorescent 2 color Whole mount in situ hybridization for a mouse embryo
https://doi.org/10.1038/protex.2013.002
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New developed fluorescent 2 color Whole mount in situ hybridization produces a clear and high S/N ratio image to analyze mRNA localization inside 1 cell and in tissue levels.
in situ hybridization
fluorescence
Whole mount in situ hybridization is a highly developed and common technique to reveal localizations of target mRNA in embryos 1) . Although it can roughly tell us localization of mRNA in tissue levels, sometimes we need more precise information for its localization, especially inside one cell of tissues. Recently, many researchers are trying to stain mRNA with fluorescent reagents with whole embryos because fluorescence have higher resolutions than chromogenic substrate and it would tell us localizations of mRNA in 1 cell level. Some reports that used fluorescence methods gave us beautiful images and suggest important conclusions from localization patterns of mRNA inside cells. However, Whole mount fluorescence in situ hybridization is sometimes very difficult for researchers, because they have to modify the protocol for each species and each embryonic stages, if they want to take a beautiful image that have high S/N ratio. In this protocol, we describe how to perform fluorescent in situ hybridization in a mouse embryo. This protocol is originally based on Zebrafish fluorescence in situ hybridization method 2) , but a probe label is different from original one to achieve clearer image3). This protocol includes tips that reduce fluorescent background and you can adopt this method to Zebrafish and other animals also.
5mg/ml ProK
2M Tris pH7.5
3M Sodium acetate
10x DIG(Roche)
RNase inhibitor
T3( Sp6 or T7) reverse transcriptase
5x reverse transcription buffer (Roche Dig labeling mix)
0.5M EDTA (pH 8.0)
8M LiCl
dNTP(ATP, CTP, GTP, UTP)
DNase I (10u/μl)
G50 Spin column (Roche)
DNP label (Mirus Labeling kit)
DEPC treated PBS.
DEPC treated PBT (DEPC treated PBS including 0.1% Tween20)
25% GlutarAldehyde
30% NH3 solution
Sodium Borohydride(SIGMA)
Formamide
10%SDS
20xSSC (pH5)
YeastRNA
Heparine
10xTBST ( 1.4M NaCl, 27mM KCL, 0.25M TrisHCl pH7.5, 1%Tween20)
DNP labeled Probe (refer to “making DNP labeled probe” )
Molecular Probe TSA kit (Alexa488 and Alexa555)
Perkin Elmer TSA Plus DNP (HRP) system
Anti-Dig-HRP (Roche)
Blocking powder (Perkin Elmer)
Hybridization oven
Water bath
Procedure
Day 1
Digest the plasmids with restriction enzyme which generate 5’ protruding terminus DNA 8μg, 10xBuffer 10μl, H2O 78μl, Enzyme 2μl (2hr) and 2μl (2hr)
Check the 1μl reacted DNA in a gel and confirm a complete digestion.
Add 4μl of 5mg/ml ProK and 10μl of 10%SDS, then incubate for 30 min at 37℃
Add 2M Tris pH7.5 6μl and phenol-chloroform 100μl, vortex and centrifuge for 5 min at room temperature with15,000 rpm.
Recover supernatant to new 1.5ml tube and then repeat step 4.
Recover supernatant to new tube and add 100μl chlorofolm. Vortex and centrifuge for 5 min at room temperature with15,000 rpm.
Recover supernatant to new tube and add 300μl Ethanol and 3M Sodium acetate
Mix with inverting a tube and centrifuge for 10 min at 4℃ with 15000rpm.
Discard supernatant and wash pellet with 80% Ethanol.
Discard supernatant and dry the pellet. Dissolve 15μl Tris-EDTA and store -20℃.
Day 2 (Ways to make a Dig probe or DNP probe are different)
For Dig labeled RNA probes
5x buffer 4μl
10x DIG 2μl
H2O 10μl (not treated with DEPC)
RNase inhibitor 1μl ( 40 Unit )
T3 or T7 1μl (10Unit,use after diluting with RNA dilution buffer )
Total; 20 μl Check the synthesized quantity 1hr after reaction by a gel running. If the quantity is small, add a little more enzyme.
After 3hrs, add following solutions directly into a reaction tube. 0.5M EDTA (8.0) 1μl
8M LiCl 1μl
EtOH 75 μl
EtOH 75 μl
For DNP labeled RNA probes
5×buffer(Roche Dig labeling mix) 12μl
ATP (Ambion, final =1mM) 6μl
CTP 6μl
GTP 6μl
UTP 6μl
H2O 12μl
Rnase Inhibitor 3μl
T7 or T3 3μl
H2O 33μl
10×Buffer A 5μl
AS 5μl
Label IT Reagent 7μl
Day 3
Dissect the embryos out in DEPC-PBS and Fix in 4% PFA.
Rocking at 4℃ for 5hrs to overnight.
Wash for 5min each with 25,50, 75% MeOH in PBT, then twice with 100%MeOH.
Rehydrate through this methanol/PBT series in reverse and wash twice with PBT.
Bleach with 6% hydrogen peroxidase in PBT for 1hr.
Wash the embryos with PBT three times for 5min.
Tread with 10μg/ml ProteinaseK in PBT for 15minutes.
Refix the embryos with 0.2% glutaraldehyde/ 4% PFA
Wash 3 times with PBT
Soak in 0.25% NH3 / 70% EtOH 20min 11.PBT wash 3times
Soak in freshly prepared 0.1% Sodium Borohydride / PBT solution for 30min and repeat this step 4 times.
PBT wash 3times
Change to PreHybridization Mix solution 2 times(50% Formamide, 5xSSC pH5, 50μg/ml Yeast RNA, 1%SDS, 50μg/ml Heparin)
PreHybridization for 30min. at 70℃ in Hybridization oven
Change solution to Hybridiztion Mix (probeA,DIG; 1μl + probeB, DNP;1μl / Prehybridization Mix solution 500μl)
Incubate overnight at 70℃
Day 4
Day 5
12.Change to blocking solution including Anti-DNP-HRP (PerkinElmer) 1μl and incubate at 4℃ for overnight with rocking
Day 6
Repeat of step 1-10 of Day 5. Tyramide should be changed to Tyramide-Alexa488.
At first, you must confirm expression patterns of mRNA by chromogenic method. If you can not obtain high S/N ratio with chromogenic ISH, it would be difficult to confirm signals by fluorescence ISH.
When background signal is high, confirm Tetrahydroborate step. Thick and large tissue might produce high background and you should cut tissues into small pieces.
You can see green and red fluorescence from each probes.
Wilkinson, D. G. In situ hybridization. A practical approach. 1992 pp. xvii + 163 pp.
Horikawa K, Ishimatsu K, Yoshimoto E, Kondo S, Takeda H. Noise-resistant and synchronized oscillation of the segmentation clock. Nature 441, 719-23 (2006).
Tetsuya Nakamura, Daisuke Saito, Aiko Kawasumi, Kyosuke Shinohara, Yasuko Asai, Katsuyoshi Takaoka, Fenglan Dong, Atsuko Takamatsu, Jose Antonio Belo, Atsushi Mochizuki, and Hiroshi Hamada. Fluid Flow and Interlinked Feedback Loops Establish Left-Right Asymmetric Decay of Cerl2 mRNA in the Mouse Embryo Nature Communications 3, 1322 (2012) doi:10.1038/ncomms2319
We thank K.Horikawa for sharing Zebrafish fluorescence ISH protocol. We thank also C.Meno, K.Yashiro and M.Yamamoto for critical advice to improve a method. This study was supported by a Grant-in-Aid for Scientific Research on Innovative Areas and a Grant-in-Aid for Young Scientists (B) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan; by Core Research for Evolutional Science and Technology (CREST) of the Japan Science and Technology Corporation (JST); and by GCOE of Osaka University.
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