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Two-color fluorescent in situ hybridization in the embryonic zebrafish brain using differential detection systems.

Lauter G, Söll I, Hauptmann G - BMC Dev. Biol. (2011)

Bottom Line: The application of different detection systems allowed for a one-step antibody detection procedure for visualization of transcripts, which significantly reduced working steps and hands-on time shortening the protocol by one day.Our protocol thus provides a novel alternative for comparison of two different gene expression patterns in the embryonic zebrafish brain at a cellular level.The principles of our method were developed for use in zebrafish but may be easily included in whole-mount FISH protocols of other model organisms.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biosciences and Nutrition, Karolinska Institutet, SE-141 83 Huddinge, Sweden.

ABSTRACT

Background: Whole-mount in situ hybridization (WISH) is extensively used to characterize gene expression patterns in developing and adult brain and other tissues. To obtain an idea whether a novel gene might be involved in specification of a distinct brain subdivision, nucleus or neuronal lineage, it is often useful to correlate its expression with that of a known regional or neuronal marker gene. Two-color fluorescent in situ hybridization (FISH) can be used to compare different transcript distributions at cellular resolution. Conventional two-color FISH protocols require two separate rounds of horseradish peroxidase (POD)-based transcript detection, which involves tyramide signal amplification (TSA) and inactivation of the first applied antibody-enzyme conjugate before the second detection round.

Results: We show here that the alkaline phosphatase (AP) substrates Fast Red and Fast Blue can be used for chromogenic as well as fluorescent visualization of transcripts. To achieve high signal intensities we optimized embryo permeabilization properties by hydrogen peroxide treatment and hybridization conditions by application of the viscosity-increasing polymer dextran sulfate. The obtained signal enhancement allowed us to develop a sensitive two-color FISH protocol by combining AP and POD reporter systems. We show that the combination of AP-Fast Blue and POD-TSA-carboxyfluorescein (FAM) detection provides a powerful tool for simultaneous fluorescent visualization of two different transcripts in the zebrafish brain. The application of different detection systems allowed for a one-step antibody detection procedure for visualization of transcripts, which significantly reduced working steps and hands-on time shortening the protocol by one day. Inactivation of the first applied reporter enzyme became unnecessary, so that false-positive detection of co-localization by insufficient inactivation, a problem of conventional two-color FISH, could be eliminated.

Conclusion: Since POD activity is rather quickly quenched by substrate excess, less abundant transcripts can often not be efficiently visualized even when applying TSA. The use of AP-Fast Blue fluorescent detection may provide a helpful alternative for fluorescent transcript visualization, as the AP reaction can proceed for extended times with a high signal-to-noise ratio. Our protocol thus provides a novel alternative for comparison of two different gene expression patterns in the embryonic zebrafish brain at a cellular level. The principles of our method were developed for use in zebrafish but may be easily included in whole-mount FISH protocols of other model organisms.

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Control of bleed-through between Fast Blue and TSA-FAM detection channels. 24-hpf embryos were hybridized to a dinitrophenol-labeled pax6a probe (A,B) or to a digoxigenin-labeled antisense RNA probe specific for nkx6.1 (C,D). Transcripts were detected using Fast Blue (A,B) and TSA-FAM (C,D). Fluorescence signals were recorded in the Fast Blue (Ch01: detection of wavelengths greater than 650 nm) and TSA-FAM (Ch02: detection of wavelengths from 505 nm to 545 nm) detection channels. No bleed-through was detected between the TSA-FAM and Fast Blue detection channels (B,C). Lateral views with anterior to the left are shown. Images were recorded on a LSM510 confocal microscope and false-colored in ImageJ. Scale bar is 50 μm.
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Figure 5: Control of bleed-through between Fast Blue and TSA-FAM detection channels. 24-hpf embryos were hybridized to a dinitrophenol-labeled pax6a probe (A,B) or to a digoxigenin-labeled antisense RNA probe specific for nkx6.1 (C,D). Transcripts were detected using Fast Blue (A,B) and TSA-FAM (C,D). Fluorescence signals were recorded in the Fast Blue (Ch01: detection of wavelengths greater than 650 nm) and TSA-FAM (Ch02: detection of wavelengths from 505 nm to 545 nm) detection channels. No bleed-through was detected between the TSA-FAM and Fast Blue detection channels (B,C). Lateral views with anterior to the left are shown. Images were recorded on a LSM510 confocal microscope and false-colored in ImageJ. Scale bar is 50 μm.

Mentions: There was no bleed-through observed, however, between the Fast Blue and TSA-FAM detection channels (Channel01: wavelengths greater than 650 nm were collected; Channel02: wavelengths between 505 nm and 545 nm were collected). Detection of pax6a by Fast Blue revealed a specific signal in the appropriate (Ch01; Figure 5A) but not in the TSA-FAM detection channel (Ch02; Figure 5B). Despite a very strong nkx6.1 TSA-FAM signal was visualized (Ch02; Figure 5D), it could not be detected in the Fast Blue detection channel (Ch01; Figure 5C). These results demonstrated the versatility of combining Fast Blue and TSA-FAM for two-color FISH.


Two-color fluorescent in situ hybridization in the embryonic zebrafish brain using differential detection systems.

Lauter G, Söll I, Hauptmann G - BMC Dev. Biol. (2011)

Control of bleed-through between Fast Blue and TSA-FAM detection channels. 24-hpf embryos were hybridized to a dinitrophenol-labeled pax6a probe (A,B) or to a digoxigenin-labeled antisense RNA probe specific for nkx6.1 (C,D). Transcripts were detected using Fast Blue (A,B) and TSA-FAM (C,D). Fluorescence signals were recorded in the Fast Blue (Ch01: detection of wavelengths greater than 650 nm) and TSA-FAM (Ch02: detection of wavelengths from 505 nm to 545 nm) detection channels. No bleed-through was detected between the TSA-FAM and Fast Blue detection channels (B,C). Lateral views with anterior to the left are shown. Images were recorded on a LSM510 confocal microscope and false-colored in ImageJ. Scale bar is 50 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3141750&req=5

Figure 5: Control of bleed-through between Fast Blue and TSA-FAM detection channels. 24-hpf embryos were hybridized to a dinitrophenol-labeled pax6a probe (A,B) or to a digoxigenin-labeled antisense RNA probe specific for nkx6.1 (C,D). Transcripts were detected using Fast Blue (A,B) and TSA-FAM (C,D). Fluorescence signals were recorded in the Fast Blue (Ch01: detection of wavelengths greater than 650 nm) and TSA-FAM (Ch02: detection of wavelengths from 505 nm to 545 nm) detection channels. No bleed-through was detected between the TSA-FAM and Fast Blue detection channels (B,C). Lateral views with anterior to the left are shown. Images were recorded on a LSM510 confocal microscope and false-colored in ImageJ. Scale bar is 50 μm.
Mentions: There was no bleed-through observed, however, between the Fast Blue and TSA-FAM detection channels (Channel01: wavelengths greater than 650 nm were collected; Channel02: wavelengths between 505 nm and 545 nm were collected). Detection of pax6a by Fast Blue revealed a specific signal in the appropriate (Ch01; Figure 5A) but not in the TSA-FAM detection channel (Ch02; Figure 5B). Despite a very strong nkx6.1 TSA-FAM signal was visualized (Ch02; Figure 5D), it could not be detected in the Fast Blue detection channel (Ch01; Figure 5C). These results demonstrated the versatility of combining Fast Blue and TSA-FAM for two-color FISH.

Bottom Line: The application of different detection systems allowed for a one-step antibody detection procedure for visualization of transcripts, which significantly reduced working steps and hands-on time shortening the protocol by one day.Our protocol thus provides a novel alternative for comparison of two different gene expression patterns in the embryonic zebrafish brain at a cellular level.The principles of our method were developed for use in zebrafish but may be easily included in whole-mount FISH protocols of other model organisms.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biosciences and Nutrition, Karolinska Institutet, SE-141 83 Huddinge, Sweden.

ABSTRACT

Background: Whole-mount in situ hybridization (WISH) is extensively used to characterize gene expression patterns in developing and adult brain and other tissues. To obtain an idea whether a novel gene might be involved in specification of a distinct brain subdivision, nucleus or neuronal lineage, it is often useful to correlate its expression with that of a known regional or neuronal marker gene. Two-color fluorescent in situ hybridization (FISH) can be used to compare different transcript distributions at cellular resolution. Conventional two-color FISH protocols require two separate rounds of horseradish peroxidase (POD)-based transcript detection, which involves tyramide signal amplification (TSA) and inactivation of the first applied antibody-enzyme conjugate before the second detection round.

Results: We show here that the alkaline phosphatase (AP) substrates Fast Red and Fast Blue can be used for chromogenic as well as fluorescent visualization of transcripts. To achieve high signal intensities we optimized embryo permeabilization properties by hydrogen peroxide treatment and hybridization conditions by application of the viscosity-increasing polymer dextran sulfate. The obtained signal enhancement allowed us to develop a sensitive two-color FISH protocol by combining AP and POD reporter systems. We show that the combination of AP-Fast Blue and POD-TSA-carboxyfluorescein (FAM) detection provides a powerful tool for simultaneous fluorescent visualization of two different transcripts in the zebrafish brain. The application of different detection systems allowed for a one-step antibody detection procedure for visualization of transcripts, which significantly reduced working steps and hands-on time shortening the protocol by one day. Inactivation of the first applied reporter enzyme became unnecessary, so that false-positive detection of co-localization by insufficient inactivation, a problem of conventional two-color FISH, could be eliminated.

Conclusion: Since POD activity is rather quickly quenched by substrate excess, less abundant transcripts can often not be efficiently visualized even when applying TSA. The use of AP-Fast Blue fluorescent detection may provide a helpful alternative for fluorescent transcript visualization, as the AP reaction can proceed for extended times with a high signal-to-noise ratio. Our protocol thus provides a novel alternative for comparison of two different gene expression patterns in the embryonic zebrafish brain at a cellular level. The principles of our method were developed for use in zebrafish but may be easily included in whole-mount FISH protocols of other model organisms.

Show MeSH