Fluorescent 2 color Whole mount in situ hybridization for a mouse embryo

doi:10.1038/protex.2013.002

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.

Cell biology

Developmental biology

Biological techniques

Molecular Biology

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

Mix following solutions and incubate at 37℃ for 3hrs. DNA(template) 2μl

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

Mix well and immediately centrifuge at 15000rpm for 10min at 4℃
80% EtOH wash
dry and dissolve in 20μl TE
add following solutions 8M LiCl 1μl

EtOH 75 μl

. Mix well and immediately centrifuge at 15000rpm for 10min at 4℃
80% EtOH wash
Dry and dissolve in DPEC H₂O 20μl
Store at -80 ℃

For DNP labeled RNA probes

Synthesis of Anti Sense probe ; Mix following solution and incubate at 37℃ 3hrs Template DNA(prepared on Day1) 6μl

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

Add 6μl DNase I (10u/μl) 6μl and incubate for 15min at 37℃
Purify RNA probes by Roche G50 column
Add 3 volumes EtOH and 3M Sodium Acetate and mix well
Centrifuge at 4℃, 15000 rpm for 10min
Discard supernatant and wash pellet by 80%EtOH.
Repeat Step5 and 6
Dry a pellet and dissolve in 20μl H₂O.
DNP label (Mirus Labeling kit) ; Mix following solutions and incubate at 37℃ for 2hrs.

H₂O 33μl

10×Buffer A 5μl

AS 5μl

Label IT Reagent 7μl

Purify RNA probe by a column(included in Miruskit)
Add 3 volumes EtOH and 3M Sodium Acetate and mix well
Centrifuge at 4℃, 15000 rpm for 10min
Discard supernatant and wash pellet by 80%EtOH.
Dry a pellet and dissolve in 5μl H₂O.

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

Wash the embryos twice with solution I (50% Formamide, 5xSSC pH5, 1%SDS) for 30min at 72℃.
Wash the embryos twice with solution III (50% Formamide, 2xSSC pH5, 0.1%Tween20) for 30 min at 72℃.
Wash 3 times for 5min with solution II (0.5MNacl, 10mMTris-HCl pH7.5, 0.1%Tween20 )
Wash embryos with 100 μg/ml RNaseA in solution II for 30 min at 37℃.
Wash the embryos for 5 min with solution II, then with solution III.
Wash twice with solution III for 30min at 67℃.
TBST wash 3 times
Change to blocking solution (0.1 M TrisHCl, pH 7.5,  0.15 M NaCl,  0.5% Blocking powder) and incubate at 4℃ with rocking for 1hr
Change to blocking solution 1ml including anti-DIG-HRP(Roche) 5μl, then incubate with rocking at 4℃ overnight.

Day 5

Wash 3 times for 5 min with TBST
TBST wash 1hr at 4℃ with rocking and repeat 5 times
Change to Amplification Buffer (Molecular Probe TSA kit.) then wait for a few minutes
Changes to Amplification Buffer 1ml including Tyramide-Alexa555 (Molecular Probe TSA kit)1μl, then incubate at 4℃ for 30min in dark room.
Make 30% H₂O₂ 1μl in Amplification Buffer 200μl (solutionA)
Make Tyramide-Alexa555 1μl, solutionA 1μl and Amplification Buffer 100μl (solutionB)
Change to solutionB and incubate for 30 min at room temperature in a dark room
TBST wash 3 times for 5min.
Change to H₂O including 0.1M Glysin, pH2.5 (pH should be adjusted by 30%HCl) and incubate for 30 min at room temperature
TBST wash 3 times for 5 min. 11.Change to blocking solution and incubate at 4℃ for 1hr with rocking

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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Fluorescent 2 color Whole mount in situ hybridization for a mouse embryo

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