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Development, Growth and Differentiation, in press (Feb. 2016)

Multi-color FISH facilitates analysis of cell-type diversification anddevelopmental gene regulation in the Parasteatoda spider embryo
Yasuko Akiyama-Oda1,2* & Hiroki Oda1
1JT Biohistory Research Hall, 1-1 Murasakicho, Takatsuki, Osaka, 569-1125 Japan

2

Microbiology and Infection Control, Osaka Medical College, Takatsuki, Osaka, Japan*To whom correspondence should be addressed.
Materials and methods
Probe synthesis
Four types of labeled antisense RNA probes, digoxigenin (DIG)-, fluorescein (FLU)-,biotin-, and 2,4-dinitrophenyl (DNP)-labeled probes, were prepared by in vitrotranscription. The former three were synthesized using 10× RNA labeling mixes, eachof which contained a modified UTP, DIG-11-UTP, FLU-12-UTP, or biotin-16-UTP(Roche 1277073, 1685619, and 1685597). DNP-labeled probes were synthesized using
DNP-11-UTP (PerkinElmer NEL555001EA) at a concentration of 0.14 mM in thetranscription reaction solution, which was 40% of the concentration of other modifiedUTPs, because a higher concentration of DNP-11-UTP tends to result in a lower probeyield. T7 and SP6 RNA polymerase-plus (Ambion AM2716 and AM2701) were used in
the transcription reaction. The cDNA templates that were used for probe synthesis wereas follows: At-fork head (fkh), At-orthodenticle (otd), (Akiyama-Oda and Oda 2003),
At-twist (twi) (Yamazaki et al. 2005), At-short gastrulation (sog) (Akiyama-Oda & Oda
2006), At-Delta (Delta), At-hedgehog (hh) (Oda et al. 2007), At-labial (lab), 022_P10,012_A08 (Akiyama-Oda & Oda, 2010), At-odd paired (opa) (Kanayama et al. 2011),
and sox2 (FY220555|At_eW_012_P20).
Embryo fixation
Spider embryos were fixed as previously described (Akiyama-Oda & Oda 2003) withsome modifications. One modification entailed the piercing of vitelline membranes witha glass capillary to make small holes. This piercing was performed in Halocarbon oil
700 (Sigma H8898) after the embryos were dechorionated with 100% commercial
bleach, washed with water and covered with oil, and this step led to the prompt
permeation of the fixative. Piercing was followed by thoroughly washing the oil with1http://www.brh2.jp/

heptane and transferring the embryos into a two-phase fixative (heptane and 5.5%
formaldehyde in PEMS (100 mM piperazine-1,2-bis(2-ethanesulfonic acid) (PIPES), 1
mM ethylenediaminetetraacetic acid (EDTA), and 2 mM MgSO4, pH 6.9)). The second
modification entailed the adjustment of the fixation time to 45-50 minutes, as a longer
fixation time leads to greater background fluorescence in the yolk. The third
modification was the use of PEMS + 0.1% Polyoxyethylene (20) sorbitan monolaurate(Tween-20) to wash the fixative, and in this solution the vitelline membranes wereremoved with forceps before gradual replacement with methanol, followed by ethanol.
The embryos were then stored in ethanol at -20°C until used for hybridization.
In situ hybridization and detection
The pretreatment, hybridization, and washing of excessive probes were performed in asimilar manner as for Drosophila embryos (Lehmann & Tautz 1994), with theexception of the following two points. First, in the pretreatment steps, H2O2 treatment(20 minutes of incubation with 2% H2O2 in methanol) was added to inactivate
endogenous peroxidase before rehydration with phosphate-buffered saline + 0.1%
Tween-20 (PBT). Second, in prehybridization and hybridization solutions, dextran
sulfate sodium salt (Sigma-Aldrich D8906) was added at a concentration of 50 µg/mL.
These two changes were performed according to Lauter et al. (2011a). The contents ofthe prehybridization and hybridization solutions were as follows: 50% formamide; 5×standard sodium citrate (SSC); 0.1% Tween-20; 50 µg/mL heparin; 100 µg/mL salmon
sperm DNA (Invitrogen 15632-011); and 50 µg/mL dextran sulfate sodium salt. In thehybridization solution, a maximum of four differently labeled RNA probes wereincluded.
After overnight hybridization, excess probes were washed with a washsolution (50% formamide, 5× SSC, 0.1% Tween-20, and 50 µg/mL heparin), followed
by gradual replacement with PBT and then with Tris-NaCl-Tween (TNT) buffer (0.1 M
Tris-HCl (pH 7.5), 0.15 M NaCl, and 0.05% Tween-20). Embryos were then blockedwith Tris-NaCl-Blocking (TNB) buffer (0.5% blocking reagent (PerkinElmer FP1020)in 0.1 M Tris-HCl (pH 7.5) and 0.15 M NaCl) for 30 minutes. This step was followed
by incubation with an antibody or with streptavidin diluted with TNB buffer for 1.5
hours at room temperature (approximately 25°C). The antibodies that were used were as
follows: anti-DIG-POD (Roche 1633716) at a dilution of 1:2000; anti-Fluorescein-HRP2

http://www.brh2.jp/(PerkinElmer NEF710001EA) at a dilution of 1:400; and anti-DNP-HRP (PerkinElmerFP1129) at a dilution of 1:200. Streptavidin-HRP (PerkinElmer NEF750001EA) wasused at a dilution of 1:2000 to detect biotin probes. After the incubation step, theantibody or streptavidin was washed with TNT buffer, followed by peroxidase reaction
with tyramide signal amplification (TSA) systems. The TSA reaction was performed
according to the method outlined by Lauter et al. (2011b); in brief, embryos were rinsed
twice with borate buffer (100 mM borate (pH 8.5), 0.1% Tween-20) and were thenincubated for 15 minutes (or for a variety of minutes in the experiment shown in Fig. 4)
in a TSA reaction solution (a tyramide reagent, 0.003% H2O2, 2% dextran sulfate
sodium salt, and 0.3 mg/mL 4-iodophenol (Sigma I10201) in borate buffer). A
commercial tyramide reagent, coumarin-tyramide, which was supplied in the TSA
Coumarin system (PerkinElmer NEL703001KT), was used at a dilution of 1:100. Other
tyramide reagents were synthesized in the laboratory (see below) and used at a dilutionof 1:200 or 1:400. Following the TSA reaction, embryos were washed several times
with PBT and incubated in 6% H2O2 in PBT for 10-30 minutes to inactivate peroxidase.
When multiple RNA probes were hybridized, the individual probes were detected insuccession; i.e., several rounds of blocking, antibody/streptavidin incubation,peroxidase reaction, and peroxidase inactivation steps were repeatedly performed. Somesamples were counterstained with 4′,6-diamidino- 2-phenylindole (DAPI;
Sigma-Aldrich).
Fluorescent tyramide synthesis
The following succinimidyl esters were used for tyramide reagent synthesis:
5-(and-6)-carboxyfluorescein succinimidyl ester (Molecular Probes C-1311);
DyLight488 N-hydroxysuccinimide (NHS) ester (Thermo Scientific 46402);
AlexaFluor568 NHS ester (Molecular Probes A20003); DyLight594 NHS ester(Thermo Scientific 46412); and DyLight680 NHS ester (Thermo Scientific 46418). Thetyramide reagents were synthesized according to the method of Lauter et al. (2011b). In
brief, each succinimidyl ester was dissolved in dimethylformamide (Sigma-Aldrich
D4551) at a concentration of 10 mg/mL. Tyramine hydrochloride (Sigma-Aldrich
T2879) solution was prepared at a concentration of 10 mg/mL in dimethylformamide.
This tyramine solution also contained triethylamine (Sigma-Aldrich T0886) at aconcentration of 10 µL/mL. For the fluorescent tyramide reagent synthesis reaction, the3http://www.brh2.jp/

tyramine hydrochloride and succinimidyl ester solutions were mixed at a molar ratio of
1:1.1. The synthesis reaction proceeded overnight in the dark at room temperature(approximately 25°C), after which the products were diluted to a concentration of 1
mg/mL with 100% ethanol.

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