Colorimetric and Fluorescent In Situ Hybridization Protocol

Colorimetric whole-mount in situ hybridization (WISH) and fluorescent in situ hybridization (FISH) were performed as elsewhere described [98 (link), 99 (link)]. The following DIG- (Roche), FITC- (Roche), or DNP- (Perkin Elmer) labeled riboprobes were synthesized using an in vitro transcription kit (Roche): Smed-β-catenin1/3/4, Smed-TCF1/2/3; Smed-opsin, Smed-otxA, Smed-sp6/9 [52 (link)]; Smed-tph [100 (link)]; Smed-ovo [54 (link)]; Smed-h2b [101 (link)]; Smed-th (tyrosine hydroxylase), Smed-tbh (tryptophan hydroxylase) [102 (link)], Smed-pc2 (prohormone convertase 2) [103 (link)]. Primers used for their synthesis are indicated (S1 Table). Riboprobes were finally diluted to 250 ng/μL in pre-hybridization solution, stored at -20°C, and were used at 1:500 in hybridization solution, except for sp6/9 (1:200). Samples were observed through Leica MZ16F (Leica Microsystems, Mannhiem, BW, Germany), Zeiss Stemi SV6 stereomicroscopes and a Zeiss Axiophot microscope (Zeiss, Jena, TH, Germany); images were captured with a ProgRes C3 camera from Jenoptik (Jena, TH, Germany), sCMEX 3.0 camera (Euromex, Arnhem, The Netherlands) and Leica DFC300FX camera (Leica Microsystems, Heerbrugg, CH, Switzerland). Confocal laser scanning microscopy was performed with a Leica TCS-SP2 (Leica Lasertchnik, Heidelberg, BW, Germany) adapted for an inverted microscope.

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Su H., Sureda-Gomez M., Rabaneda-Lombarte N., Gelabert M., Xie J., Wu W, & Adell T. (2017). A C-terminally truncated form of β-catenin acts as a novel regulator of Wnt/β-catenin signaling in planarians. PLoS Genetics, 13(10), e1007030.

Publication 2017

FITC- Leica MZ16F Zeiss Axiophot microscope ProgRes C3 camera Leica DFC300FX camera

Colorimetric Confocal laser scanning microscopy Fitc Fluorescent in situ hybridization Hybridization In situ hybridization Microscope Opsin Primers Prohormone convertase 2 Stemi Synthesis Tcf1 Transcription Tryptophan hydroxylase Tyrosine hydroxylase

Corresponding Organization : Universitat de Barcelona

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Variable analysis

independent variables

Colorimetric whole-mount in situ hybridization (WISH)
Fluorescent in situ hybridization (FISH)

dependent variables

Expression patterns of the following genes: Smed-β-catenin1/3/4, Smed-TCF1/2/3, Smed-opsin, Smed-otxA, Smed-sp6/9, Smed-tph, Smed-ovo, Smed-h2b, Smed-th (tyrosine hydroxylase), Smed-tbh (tryptophan hydroxylase), Smed-pc2 (prohormone convertase 2)

control variables

Riboprobe concentrations (250 ng/μL in pre-hybridization solution, used at 1:500 in hybridization solution, except for sp6/9 at 1:200)
Microscopy techniques (Leica MZ16F, Zeiss Stemi SV6 stereomicroscopes, Zeiss Axiophot microscope, Leica TCS-SP2 confocal laser scanning microscope)

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