We describe a multi-target chromogenic whole-mount in situ hybridization (MC-WISH) procedure in intact Drosophila embryos allowing simultaneous and specific detection of three different mRNA distribution patterns by contrasting color precipitates.
To analyze gene regulatory networks active during embryonic development and organogenesis it is essential to precisely define how the different genes are expressed in spatial relation to each other in situ. Multi-target chromogenic whole-mount in situ hybridization (MC-WISH) greatly facilitates the instant comparison of gene expression patterns, as it allows distinctive visualization of different mRNA species in contrasting colors in the same sample specimen. This provides the possibility to relate gene expression domains topographically to each other with high accuracy and to define unique and overlapping expression sites. In the presented protocol, we describe a MC-WISH procedure for comparing mRNA expression patterns of different genes in Drosophila embryos. Up to three RNA probes, each specific for another gene and labeled by a different hapten, are simultaneously hybridized to the embryo samples and subsequently detected by alkaline phosphatase-based colorimetric immunohistochemistry. The described procedure is detailed here for Drosophila, but works equally well with zebrafish embryos.
In situ hybridization (ISH) is the standard method for detection and localization of RNA transcripts in a morphological context, within cells, tissues and organisms 1. The signals produced by the ISH procedure are commonly visualized by radioactive, fluorescent and chromogenic detection systems. In recent years significant technological advances in fluorescent ISH (FISH)2 resulted in dramatically improved sensitivity and resolution, allowing the detection and quantitation of RNA expression at single-cell and sub-cellular levels and RNA visualization up to single molecules 3,4.While sophisticated single-molecule FISH methods are used for more specialized applications, chromogenic ISH is widespread as a routine RNA in situ detection method in research and clinical diagnostics. For chromogenic detection enzyme precipitation reactions are used, which generate visible products in contrasting colors at the sites of hybridization 5. This has the advantage that RNA visualization can be combined with routine histological stains and morphological context is immediately evident by standard brightfield microscopy. Moreover, numerous of the applied color substrates produce precipitates, which are stable in organic and/or aqueous mounting media, so that permanent sample preparations can be obtained 5.
In Drosophila, initial ISH protocols applied radioisotope-labeled probes for transcript detection on sectioned material 6. While it is difficult to reconstruct from tissue sections complete transcript patterns of entire embryos or organ systems, the application of chromogenic ISH procedures with non-radioactively labeled probes makes it possible to globally detect RNA distributions in whole-mounts 7. Although many variations of chromogenic ISH exist, in typical whole-mount in situ hybridization (WISH) protocols hybridized hapten-labeled probes are detected by anti-hapten antibodies conjugated to a reporter enzyme and mRNA transcripts are visualized by a precipitating chromogen.
The palette of differently colored precipitates produced by the reporter enzymes alkaline phosphatase (AP), horseradish peroxidase (POD), and beta-galactosidase (GAL) allows for the distinctive detection of multiple targets in one and the same sample 8-14. However, POD enzymatic activity lasts only for a limited time period and the GAL colorimetric reaction is somewhat less sensitive, so that without additional (tyramide) signal amplification 15 the detection of less abundant transcripts can be challenging with these enzymes. In contrast, the enduring activity of AP allows for long-lasting substrate turnover and high signal-to-noise ratio. Therefore, sequential detection using AP reporter enzyme with differently colored substrates has proven successful in the effective and distinctive detection of up to three different transcripts in single embryos 10-12,16.
For this multi-target chromogenic WISH (MC-WISH) method (Figure 1) 14, labeled antisense RNA probes are generated by in vitro transcription and marked with one of the available hapten-labels. Embryos are formaldehyde fixed and permeabilized by methanol treatment and proteinase K digestion. Hybridization of embryos is simultaneously carried out with up to three differently labeled antisense RNA probes each specific for a different gene. After removal of unbound probe by stringency washes each hapten-label is visualized in a separate round of detection. A single detection round consists of incubation of embryos with an anti-hapten antibody coupled to AP and RNA visualization by application of an AP-substrate that produces a localized stable color precipitate. After antibody detection and staining, the applied antibody-AP conjugate is removed by a low pH wash. In multicolor experiments, each round of detection employs an antibody targeted against a different hapten-label and each transcript pattern is visualized by a different color substrate (Table 1). Embryos are mounted in glycerol and imaged under a high-resolution compound microscope using differential interference contrast (DIC) optics.
1. Labeling of RNA probes by In Vitro Transcription
2. Processing of Embryo Samples Using Inserts
Note: Using microtubes or multi-well plates for processing embryos through the different incubation steps bears the risk of losing significant amounts of embryos by accidentally aspirating them during exchange of solutions. To avoid this loss, it may be helpful to use baskets (such as Netwell inserts) for carrying embryo samples through the procedure. These are polystyrene inserts with a polyester mesh at the bottom (Figure 2A), on which the embryos rest during processing.
3. Preparation of Embryos for Hybridization
4. Hybridization of Probes
5. Antibody Detection of Hapten-labeled Probes
Note: In contrast to the simultaneous hybridization of all three differently labeled probes, each probe is detected one after another in separate rounds of antibody incubation and staining. Therefore, in each detection round only one antibody is used (either anti-digoxigenin-AP or anti-fluorescein-AP or anti-biotin-AP).
6. Alkaline Phosphatase Chromogenic Staining
7. Antibody Removal/Alkaline Phosphatase Inactivation
8. Second Detection Round
9. Third Detection Round
10. Mounting and Imaging
The described protocol allows for the simultaneous visualization of multiple transcript patterns in different colors. MC-WISH provides the advantage to directly compare the expression patterns of different genes in the same embryo. As an example, the expression patterns of empty spiracles (ems) 19, fushi tarazu (ftz) 20 and sloppy paired 1 (slp1) 21 are directly compared in Drosophila melanogaster blastoderm stage embryos and visualized in a variety of color shades (Figure 4).
Different AP substrate combinations are available to obtain a panel of contrasting color precipitates (Table 1). The red color precipitate produced by Fast Red and the purple precipitate produced by BCIP/NBT can be easily distinguished and are therefore used most commonly in two-color experiments (Figure 4A). However, the BCIP/NBT stain can quickly turn into a rather dark color, so that the Fast Red color is disguised in case of partial co-distribution of transcripts. Therefore, the application of Fast Blue can be of advantage, which generates blue, green and violet products in combination with NAMP, NAGP and NABP, respectively (Table 1). The combination of Fast Blue with NAMP gives a light blue color that is easily discernible from the Fast Red product in adjacent or overlapping expression domains as shown here for ems and slp1 expression sites visualized in red and blue, respectively (Figure 4B). The Fast Blue NAGP combination provides a similar strong contrast to the Fast Red stain, so that the green ems domain is clearly distinctive from the red slp1 expression site (Figure 4C). However, if ems and slp1 expression sites are detected by Fast Blue NABP and Fast Red, respectively, the resulting violet and red color precipitates are less discernible (Figure 4D). As mentioned above, Fast Red and Fast Blue NAGP are easy to distinguish as shown by slp1 and ftz expression in red and green, respectively (Figure 4D).
MagentaPhos in combination with INT produces a yellow precipitate, which is used to reveal the segmental expression of ftz (Figure 4C, E). The yellow colored precipitate generates good contrast to the blue colored substrates as exemplified by the detection of slp1 and ftz with Fast Blue NAMP and MAG/INT, respectively (Figure 4E). However, the yellow precipitate is less easy discernible from the Fast Red product. The yellow stained expression domain of ems can be hardly distinguished from the rostral expression of slp1 shown in red (Figure 4F). Therefore, the combination of color substrates has to be carefully chosen for each experiment.
Figure 1. Flowchart of MC-WISH. For multicolored visualization of three unique transcript patterns, choose between different antibody-AP conjugates and color substrate combinations in each detection cycle. Abbreviations: AP, alkaline phosphatase; BIO, biotin; DIG, digoxigenin; FLUO, fluorescein. For other abbreviations see legends to Figure 4 and Table 1. Please click here to view a larger version of this figure.
Figure 2. Inserts as embryo carriers. (A) Inserts fitted at the bottom with polyester membranes of 15 mm diameter and 74 µm mesh size are used as Drosophila embryo carriers. (B) The inserts are assembled in 12-well plates, which function as reservoir trays for the various solutions. (C) Available carriers and handles allow processing of (D) 12 inserts/embryo samples simultaneously. Please click here to view a larger version of this figure.
Figure 3. Mounting of stained embryos. Embryos are mounted in a drop of 70% glycerol between two stacks of coverslips, which function as spacers. Gentle moving of the applied large coverslip helps to rotate the mounted embryos into the desired orientation for observation and photography. Please click here to view a larger version of this figure.
Figure 4. Multicolor visualization of mRNA transcripts in Drosophila embryos. Examples of MC-WISH experiments in Drosophila embryos are shown from lateral views. The mRNA expression patterns of empty spiracles (ems), fushi tarazu (ftz) and sloppy paired 1 (slp1) are visualized by different AP color substrate combinations, which are indicated on each panel. Transcripts were detected by (A) ftz FLUO and ems DIG, (B) slp1 FLUO, ems BIO, and ftz DIG, (C) ems BIO, slp1 FLUO, and ftz DIG, (D) ftz BIO, slp1 FLUO, and ems DIG, (E) slp1 FLUO, ems DIG, and ftz BIO, (F) ftz BIO, slp1 FLUO, and ems DIG labeled probes. The hapten-labeled probes were immunohistochemically detected one after another in the listed orders. Abbreviations: FB Fast Blue; FR, Fast Red tablet. For other abbreviations see legends to Figure 1 and Table 1. Please click here to view a larger version of this figure.
Substrate combination | Add to 1 ml AP buffer | Concentration [µg/ml] | AP buffer | Color | Signal sensitivity |
BCIP NBT | 3.5 µl 4.5 µl | 175 337.5 | SB9.5 | purple | +++ |
MAG INT | 5 µl 5 µl | 250 250 | SB9.5 | yellow | + |
BCIP/INT stock solution | 7.5 µl | 250 250 | SB9.5 | yellow | + |
Fast Blue NAMP | 5 µl 5 µl | 250 250 | SB8.2 | blue | ++ |
Fast Blue NABP | 20 µl 10 µl | 1,000 500 | SB8.2 | violet | ++ |
Fast Blue NAGP | 10 µl 10 µl | 500 500 | TT8.2 | green | + |
Fast Red NAMP | 1 tablet | 1,000 400 | TT8.2 | red | ++ |
Table 1. Substrate combinations. Abbreviations: +++, strong; ++, medium; +, weak NBT, 4-nitro-blue-tetrazolium chloride; BCIP, 5-bromo-4-chloro-3-indolyl-phosphate; INT, 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-tetrazolium chloride; MAG, Magenta-Phos, 5-bromo-6-chloro-3-indolyl-phosphate, NAMP, Naphthol-AS-MX-phosphate; NABP, Naphthol-AS-BI-phosphate; NAGP, Naphthol-AS-GR-phosphate; SB9.5, staining buffer at pH 9.5; SB8.2, staining buffer at pH 8.2; TT8.2, Tris-Tween buffer at pH 8.2.
In situ hybridization (ISH) with radioactively labeled nucleic acid probes is often used to detect the localization of RNA on tissue sections. The radioactive ISH method, however, is time consuming, less sensitive, and does not allow appreciation of complete transcript distribution patterns in whole-mounts. In contrast, the herein described MC-WISH method permits the direct comparison of multiple gene expression domains in contrasting colors within intact embryos. MC-WISH has the advantage that histological context is immediately evident and visualized simply by standard brightfield microscopy. This provides the possibility to relate gene expression domains topographically to each other with high accuracy and define unique and overlapping expression sites in a fast and reliable way. On the other hand, multiplexed fluorescent ISH (FISH) is more powerful with respect to sensitivity and resolution and is preferably used, if cellular or even sub-cellular resolution of mRNA detection is required.
The wide spectrum of transcripts that has been mapped by MC-WISH suggests that virtually any transcript can be analyzed not only in embryonic but also in larval and adult tissues and in species other than Drosophila. Indeed, the described procedure detailed here for Drosophila, works similarly efficient with zebrafish embryos 11,16,22. Moreover, the MC-WISH method can be adapted to a wide variety of invertebrate and vertebrate embryos and tissue specimen.
Hapten-labels and probe concentrations
We routinely use fluorescein, digoxigenin, and biotin as hapten labels of RNA probes. Moreover, dinitrophenol-labeled probes may be applied, which have been introduced in zebrafish WISH experiments 23-25. Fluorescein-labeled probes display a lesser sensitivity in comparison to the other hapten-labels, so that fluorescein is best used for detection of abundant transcripts. Each newly transcribed probe is first tested in a single WISH experiment, where its performance is evaluated either using BCIP/NBT or Fast Red as substrate. A probe concentration is considered as optimal when a strong signal without background development is achieved within min to few hr.
To make sure that the expression domains of interest have been detected in their full extent by MC-WISH, it is essential to visualize each transcript pattern by single-label WISH experiments using the most sensitive substrate combination, BCIP/NBT. This ensures that weak expression domains are not overlooked in the multi-target experiment. To compensate for the lesser sensitivity of the other substrate combinations it is essential to use doubled (to tripled) probe concentrations as compared to standard BCIP/NBT staining.
Low pH inactivation
The low pH inactivation step can lead to partial disintegration of the antisense hapten-labeled RNA probe/sense mRNA hybrids resulting in reduced signal detection in the second and third staining rounds. To minimize loss in sensitivity, the inactivation steps are as short as possible. We experienced that a 10 min incubation time at low pH is sufficient for elimination of AP-activity. The subsequent quick washes in large volumes ensure rapid dilution of unbound antibody-AP conjugates and prevent re-binding to the haptenized probes. Alternative procedures include paraformaldehyde fixation and heat inactivation. However, some of the color precipitates are not heat stable and paraformaldehyde fixation may not be sufficient for complete inactivation of AP. Incomplete inactivation of the first applied antibody-AP conjugate can lead to re-visualization of the mRNA pattern in the following detection round leading to false positive overlap in expression with the second mRNA species to be detected. Therefore, in a control experiment the embryos are split into two fractions after inactivation of the first applied antibody-AP conjugate. The second antibody-AP conjugate is omitted from the control fraction and should not produce a signal in the second color reaction. However, if the second color reaction generates a signal distribution pattern corresponding to the first one, then the inactivation procedure was not efficient. In this case, the incubation time in low pH stop solution should be prolonged for the next experiment.
Orders of AP substrate application
Since sensitivity drops with each subsequent round of detection, it is advisable to detect less abundant mRNAs prior to more abundant transcripts. In addition, the Fast Red and Fast Blue substrate combinations are significantly less sensitive than the purpleblue BCIP/NBT stain, so that Fast dyes are preferably applied in the first staining round for detection of the stronger expressed transcript. This also has the advantage that subsequent BCIP/NBT staining can be monitored and stopped in time before the purpleblue signal gets too dark in relation to the lighter Fast dyes. Thus in a standard two-color experiment, first the stronger expressed mRNA is detected by Fast Red and second the weaker one by BCIP/NBT. As an alternative to the Fast dyes the yellow INT substrate combinations may be applied, which however produce precipitates that become diffuse after some time. Therefore BCIP/INT is exclusively applied in the last staining round. Consequently in a three-color experiment we often use first Fast Red, second BCIP/NBT (or Fast Blue) and third an INT substrate combination. Note that it is advisable to photograph stained embryos as soon as possible when applying INT.
Fluorescent detection of azo dyes
It can be sometimes difficult to recognize overlapping expression patterns when a darker color precipitate is shadowing a lighter one. For example, a strongly developed BCIP/NBT precipitate can mask lighter Fast dye signals. One way to get around this problem is to capture images immediately after each staining and remove the applied color precipitate before the next detection round. In this case, alcohol-soluble azo dye (Fast Red) and INT precipitates are removed by ethanol washes after the first and second detection rounds, respectively, and BCIP/NBT staining is applied as the last AP-substrate 26. Another possibility is to take advantage of the fluorescent properties of azo dyes. Fast Red can be visualized using rhodamine filter sets 27, while Fast Blue can be observed with far-red filters 28,29. Comparison or overlay of chromogenic and fluorescent images can reveal the site of co-distribution.Using this approach, BCIP/NBT signal development has to be stopped in time, so that it does not become too dense and quench the fluorescent signal of the azo dye reaction product.
The authors have nothing to disclose.
We thank our colleagues for cDNA plasmids for template generation. The authors’ work is supported by the Swedish Cancer Foundation (CAN 2010/553), the Swedish Foundation for International Cooperation in Research and Higher Education (IG2011-2042) and the Knut and Alice Wallenberg Foundation (KAW2012.0058).
Deionized, diethylpyrocarbonate (DEPC) treated water | Thermo Scientific | R0603 | |
T7 RNA Polymerase | Thermo Scientific | EP0111 | |
T3 RNA Polymerase | Thermo Scientific | EP0101 | |
SP6 RNA Polymerase | Thermo Scientific | EP0131 | |
RiboLock RNase Inhibitor | Thermo Scientific | EO0381 | |
DNase I, RNase-free | Thermo Scientific | EN0521 | |
NTP Set | Thermo Scientific | R0481 | |
Digoxigenin-11-UTP | Roche | 11209256910 | |
Fluorescein-12-UTP | Roche | 11427857910 | |
Biotin-16-UTP | Roche | 11388908910 | |
Ammonium acetate | Applichem | A2936 | |
Ethanol, absolute | Merck | 100983 | |
di-Sodium hydrogen phosphate | Scharlau | SO0339 | |
Sodium dihydrogen phosphate | Scharlau | SO0331 | |
Tween-20 | Sigma | P1379 | |
Paraformaldehyde | Sigma | P6148 | |
Methanol | Sigma | 32213 | |
Proteinase K | Thermo Scientific | EO0491 | |
Glycine | Sigma | G7126 | |
Hydrochloric acid | Merck | 100317 | |
tri-Sodium citrate | Scharlau | SO0200 | |
deionized formamide | Applichem | A2156 | |
RNA type VI from torula yeast | Sigma | R6625 | |
Heparin sodium salt | Applichem | A3004 | |
Dextran sulfate sodium salt | Sigma | D6001 | |
Sheep serum | Sigma | S2263 | |
Sheep anti-biotin-AP Fab fragments | Roche | 11426303001 | |
Sheep anti-digoxigenin-alkaline phosphatase (AP) Fab fragments | Roche | 11093274910 | |
Sheep anti-fluorescein-AP Fab fragments | Roche | 11426303001 | |
Rabbit anti-dinitrophenyl-AP antibody | Vector laboratories | MB-3100 | |
Tris-(hydroxymethyl)-aminomethane | Scharlau | TR04241000 | |
Magnesium chloride | Scharlau | MA0036 | |
Sodium chloride | Scharlau | SO0227 | |
Levamisole | Sigma | L9756 | |
N,N-Dimethylformamide | Sigma | D4551 | |
Dimethylsulfoxide | Applichem | A3006 | |
Fast Red tablet set | Sigma | F4648 | |
Fast Blue BB salt | Sigma | F3378 | |
Naphthol-AS-MX-phosphate | Sigma | N5000 | |
Naphthol-AS-GR-phosphate | Sigma | N3625 | |
Naphthol-AS-BI-phosphate | Sigma | N2250 | |
Magenta-Phos | Biosynth | B7550 | |
INT | Sigma | I8377 | |
NBT | Applichem | A1243 | |
BCIP | Applichem | A1117 | |
INT/BCIP solution | Roche | 11681460001 | |
Glycerol 86-88% | Scharlau | GL0023 | |
Universal incubator | Memmert | BE400 | |
Waterbath | Memmert | WB14 | |
Heat block | Grant | QBT2 | |
Netwell inserts 15 mm | Corning | 3477 | |
Netwell carrier kit 15 mm | Corning | 3520 | |
12-well cell culture plate | Corning | 3513 | |
24-well plate | Sarstedt | 83.3922 | |
1.5 ml microtube | Sarstedt | 72.690.001 | |
2.0 ml microtube | Sarstedt | 72.691 |