Hurtado R and Mikawa T (2006), Enhanced sensitivity and stability in two-color...

XB-ART-42732

Dev Dyn 2006 Oct 01;23510:2811-6. doi: 10.1002/dvdy.20909.

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Enhanced sensitivity and stability in two-color in situ hybridization by means of a novel chromagenic substrate combination.

Hurtado R , Mikawa T .

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Double in situ hybridization analysis is a fundamental technique for studying the expression of two genes with high temporal and spatial resolution. However, due to the lack of sensitivity in current detection methods, this approach is powerful only when at least one transcript is abundantly expressed. Here, we report a new enzyme/chromagenic substrate combination that provides sufficient sensitivity for detecting two less abundant transcripts and stability for subsequent paraffin sectioning.

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Species referenced: Xenopus
Genes referenced: fgf8 pax2 shh tbx5 tubb2b

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	Figure 1. The high sensitivity of color development reaction with FR-BCIP comparable with NBT-BCIP. Whole-mount ISH for Tbx5 mRNA in E3 chick embryos using different color substrates, NBT-BCIP (A), INT-BCIP (D), FR-Naphthol (G), and FR-BCIP (J). High magnification of the eye (B,E,H,K) and the heart (C,F,I,L). reverse transcriptase-polymerase chain reaction (PCR) analysis of Tbx-5 and GAPDH mRNA (M). The number of PCR cycles used is indicated. e, eye; h, heart; l, limb; FR, Fast Red 1-5-naphethediol; BCIP, 5-bromo-4-chloro-3indolyl-phosphate; NBT, nitroblue-tetrazolium chloride; ISH, in situ hybridization; INT, 2-(4-iodopenyl)-3-(4-nitropenyl)-5-phenyltetrazolium chloride.
	Figure 2. Higher FR concentrations diminish FR-BCIP color development. A: ISH for VMHC1 (Bisaha and Bader,1991) on embryonic day (E) 6 chick hearts varying concentration of FR at 0.06 μg/μl (A), 0.10 μg/μl (B), 0.20 μg/μl (C), and 0.27 μg/μl (D) with the constant concentration of BCIP. A,B: Note that, at lower concentrations of FR, color development was detected throughout the ventricular myocardium visualizing the known VMHC1 expression pattern in the embryonic heart at this stage. C,D: Higher concentration of FR resulted in diminished color reaction (C) associated with a high yellowish background (D). ra, right atrium; la, left atrium; rv, right ventricle; lv, left ventricle; FR, Fast Red 1-5-naphethediol; BCIP, 5-bromo-4-chloro-3indolyl-phosphate; ISH, in situ hybridization; VMHC1, ventricular myosin heavy chain-1.
	Figure 3. FR-BCIP color precipitate is distinct from that of NBT-BCIP in whole-mount DISH. A,B: Single ISH for Pax2 mRNA with NBT-BCIP. C: DISH for Pax2 mRNA with NBT-BCIP and for Tbx5 mRNA with either INT-BCIP (C,D), FR-Naphthol (E,F), or FR-BCIP (G,H). High magnification of the eye (B,D,F,H). e, eye; h, heart; hb, hind brain; l, limb; nd, nephric duct; op, otic placode. FR, Fast Red 1-5-naphethediol; BCIP, 5-bromo-4-chloro-3indolyl-phosphate; DISH, double in situ hybridization; ISH, in situ hybridization; INT, 2-(4-iodopenyl)-3-(4-nitropenyl)-5-phenyltetrazolium chloride.
	Figure 4. DISH with NBT-BCIP and FR-BCIP for Tbx5 and VMHC1 showing identifiable zones of overlapping expression. A: Single ISH for Tbx5 mRNA using FR-BCIP. Note expression (turquoise arrows) in the right atrium (ra), the left atrium (la) and the left ventricle (lv), but no detectable expression in the right ventricle (rv). B: Single ISH for VMHC1 mRNA using NBT-BCIP, showing expression in the left and right ventricles (purple arrows) but no detectable expression in the atria. C: DISH for Tbx5 and VMHC1 mRNAs. Note that overlapped expression of the two genes is evident in the left ventricle (turquoise and purple arrows), whereas no significant coexpression is detectable in the atria or right ventricle. DISH, double in situ hybridization; NBT, nitroblue-tetrazolium chloride; BCIP, 5-bromo-4-chloro-3indolyl-phosphate; FR, Fast Red 1-5-naphethediol; VMHC1, ventricular myosin heavy chain-1;
	Figure 5. Stability of FR-BCIP precipitates through post-DISH paraffin sectioning. A: DISH for Fgf8 with NBT-BCIP and Shh with either INT-BCIP (A,B), FR-Naphthol (C,D), and FR-BCIP (E,F). After whole-mount DISH (A,C,E), embryos were processed for paraffin sectioning. High-magnification view (B,D,F) of areas containing notochord and floor plate from post-DISH paraffin sections (inset). ba, branchial arches; fb, forebrain; fp, floor plate; nc, notochord; FR, Fast Red 1-5-naphethediol; BCIP, 5-bromo-4-chloro-3indolyl-phosphate; DISH, double in situ hybridization; NBT, nitroblue-tetrazolium chloride; INT, 2-(4-iodopenyl)-3-(4-nitropenyl)-5-phenyltetrazolium chloride.