PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Dev Dyn. Author manuscript; available in PMC Feb 1, 2010.
Published in final edited form as:
PMCID: PMC2640425
NIHMSID: NIHMS90533
A Formaldehyde-based Whole-Mount In Situ Hybridization Method for Planarians
Bret J. Pearson,* George T. Eisenhoffer,* Kyle A. Gurley,* Jochen C. Rink,* Diane E. Miller, and Alejandro Sánchez Alvarado
Department of Neurobiology and Anatomy Howard Hughes Medical Institute University of Utah School of Medicine 401 MREB, 20N 1900E Salt Lake City, UT 84132
*these authors contributed equally to this work
Author for correspondence: sanchez/at/neuro.utah.edu
Whole-mount in situ hybridization (WISH) is a powerful tool for visualizing gene expression patterns in specific cell and tissue types. Each model organism presents its own unique set of challenges for achieving robust and reproducible staining with cellular resolution. Here we describe a formaldehyde-based WISH method for the freshwater planarian Schmidtea mediterranea developed by systematically comparing and optimizing techniques for fixation, permeabilization, hybridization and post-processing. The new method gives robust, high-resolution labeling in fine anatomical detail, allows co-labeling with fluorescent probes, and is sufficiently sensitive to resolve the expression pattern of a microRNA in planarians. Our WISH methodology not only provides significant advancements over current protocols that make it a valuable asset for the planarian community, but should also find wide applicability in WISH methods used in other systems.
Keywords: in situ, whole mount, planaria, mucolytics
Freshwater planarians are well known for their ability to regenerate from injuries considered catastrophic in other animals. When a planarian is decapitated, for example, the resulting trunk regenerates a new head with all the attendant organs (e.g., brain, photoreceptors, sensory neurons) within a week. Even a fragment accounting for roughly 1/279th of the former body size can regenerate a complete animal (Morgan, 1898). The fact that a large population of adult somatic stem cells is required for planarian regeneration has fuelled the recent surge in interest in understanding the molecular mechanisms behind this process (Newmark and Sánchez Alvarado, 2002). A key molecular technique commonly used in planarian and other developmental biology studies is whole-mount in situ hybridization (WISH), which allows for the visualization of gene transcripts within cells and tissues. The central goal of a WISH method is to provide investigators with the technical ability to: a) define specific cell types by their gene expression pattern; b) analyze the distribution of cell types within the tissue architecture; and c) study the functional interactions between multiple cell types. However, as the need for more detailed analysis of planarian stem cells and regeneration has proceeded, so has the need for more robust and sensitive WISH methods.
A critical component of successful WISH involves balancing the often contrasting demands between sensitivity and preservation of morphology. While the retention of transcripts and preservation of overall tissue morphology necessitates use of a fixative, permeabilization of tissues and cells is a prerequisite for optimal access of antisense riboprobes to their targets. Any given organism presents its own set of challenges to achieve this balance. For example, in Drosophila embryos, the chorion and vitelline membrane must be removed in order to permeabilize, fix, and visualize expression patterns. Zebrafish embryos can be fixed in the chorion, but extensive permeabilization steps must be performed prior to WISH due to the relatively higher tissue density when compared to many invertebrates. While WISH is rarely performed in C. elegans, WISH protocols commonly use a histological fixative called Bouin's to permeabilize the tough cuticle.
Even though it has been empirically determined in the vast majority of systems that formaldehyde (FA) fixations are optimal for WISH, planarians have not been amenable to this approach. This is mainly due to two major distinguishing characteristics. First, these organisms secrete a dense mucus layer around their entire bodies thought to facilitate osmotic balance, locomotion, and predator avoidance (Hyman, 1951). The mucus layer also presents a considerable barrier to the exchange of fluids and large molecules needed during WISH. Second, experimental animals are often several millimeters in length, posing special challenges with respect to detection efficiencies in deep tissues. Previously, it was found that an alcohol/acid based fixative called Carnoy's could penetrate the mucus barrier and yield WISH signals in planarians (Umesono et al., 1997). This method quickly became the field standard and is now widely used. However, we find that the degree of staining in a batch of Carnoy's fixed animals can be highly variable. Moreover, Carnoy's is not a cross-linking fixative, and thus the balance between permeability and transcript retention is not optimal (Urieli-Shoval et al., 1992). In contrast, the cross-linking capabilities of FA-based fixations offer higher cellular resolution and thus have emerged over the last 2 decades as the preferred WISH fixative across a wide variety of model systems. The need for cellular resolution and simultaneous labeling with multiple riboprobes has promoted our attempts to develop a more sensitive and robust WISH method for planarians.
Here we report an FA-based, sensitive, and reproducible whole-mount WISH methodology for planarians. Different techniques for fixation, permeabilization, hybridization and post-processing were tested for their general utility and were systematically optimized for maximal synergy. The effects of key innovations in the protocol are demonstrated with a set of markers labeling a range of planarian cell and tissue types. We find that the new methodology is superior to Carnoy's fixation for all tested markers, allowing the visualization of fine anatomical details at cellular resolution, yet at a sensitivity level sufficient even for the detection of microRNA (miR-124a) expression. Finally, we show that double fluorescent WISH along with antibody-staining of protein-epitopes provides robust access to multi-cell-type analysis using this optimized method. Hence the method described here represents a valuable asset for the planarian community and our optimizations should be widely applicable to WISH methods in other systems.
Key optimization steps for a new planarian WISH method
WISH methods contain countless variables that can be subjected to optimization. To develop a robust and sensitive WISH method for the planarian Schmidtea mediterranea, we concentrated on the following strategic steps: killing and mucus removal, fixation, permeabilization, dehydration, bleaching, riboprobe hydrolysis, hybridization solution (Hyb) composition, and post-development treatments (Refer to “Detailed Methods” section below). To ensure the universality of our optimizations, these parameters were tested using multiple riboprobes of varying signal intensities, tissue depths, and anatomical locations.
In figure 1, six conditions using four representative riboprobes depict how key optimizations improved staining compared to Carnoy's. In order of highest to lowest staining intensity, the probes used were: 1) smedwi-1 (Reddien et al., 2005), a stem cell marker in planaria that produces intense staining deep within in the animal; 2) Smed-wnt-2 (Gurley et al., 2008), which detects cells in a posterior-to-anterior gradient (Gurley et al., 2008; Petersen and Reddien, 2008); 3) Smed-slit (Cebria et al., 2007), a marker that stains both surface and deep cells along the midline; and 4) Smed-PCNA (Eisenhoffer et al., 2008), which labels cycling stem cells. While the minimal FA fixation decreased overall signal intensity for all 4 probes relative to Carnoy's fixation, the signal obtained in FA was less diffused and resolved into a discrete, cell-specific pattern. (Fig. 1B; rows 1-2). A simple treatment with 100% ethanol for ~20min after development improved signal to noise ratios, which greatly helped determine which aspects of staining were due to nonspecific background (Fig.1B; row 3). However, smedwi-1 still stained much darker in Carnoy's fixation than FA fixation, indicating that permeabilization was sub-optimal. We therefore replaced hydrochloric acid, normally used to kill worms (Umesono et al., 1997), with the mucolytic compound N-acetyl-cysteine (NAC), which both killed the worms and removed the mucus layer surrounding the animals. NAC, along with a short SDS-permeabilization step, greatly increased staining intensity and specificity (Fig 1B; row 4).
Figure 1
Figure 1
Key steps to an optimized FA-based WISH method for planarians
Because the dehydration of specimens prior to hybridization is commonly used in many WISH protocols to increase staining intensity, we tested dehydration conditions. All 4 probes were intensified by dehydration in methanol or ethanol (Fig. 1B; row 5). Interestingly, dehydration revealed a posterior-anterior gradient of Smed-wnt-2 expression. Another commonly used method to increase mRNA accessibility for hybridization is treatment with proteinase K. We generally found that proteinase K treatment followed by a short post-fixation increased staining intensity (Fig. 1B; row 6). However, these conditions remained sub-optimal because the pre-pharyngeal region of the animals became more refractory to WISH. To penetrate these tissues, we replaced the SDS permeabilization step with a “reduction” step that utilizes the reducing agent dithiothreitol (DTT) and an additional detergent (NP40; Fig. 1B, row 7). Not only did this treatment allow staining in impermeable pre-pharyngeal regions, but it also intensified staining for each probe.
With all conditions combined, it is important to note differences with the current standard Carnoy's used in the majority of planarian publications. First, for strong probes such as smedwi-1, our protocol achieves similar staining intensity but with vastly improved cellular resolution and animal morphology. For slightly weaker probes like Smed-wnt-2 and Smed-slit, our WISH method improves on all metrics of a WISH stain: improved intensity, minimal background, increased cellular resolution, and complete and thorough staining penetration. Finally, the weak probe for Smed-PCNA shows the greatest improvements with FA vs. Carnoy's. Whereas there is high background and very little specific staining observed in Carnoy's, we achieved cellular resolution and appreciably better staining intensity with minimal background using the Smed-PCNA riboprobe in our optimized FA method.
Optimizations also improved Carnoy's fixations, yet FA fixations increased cellular resolution and higher signal to noise ratios
To test whether our optimizations also improved staining for Carnoy's fixation, we used NAC treatment, ethanol post-processing, and optimized hybridization buffer on Carnoy's fixed animals. We assumed that the proteinase K and reduction steps would not help because Carnoy's-treated animals are already highly-permeable. In addition, Carnoy's does not covalently cross-link molecules and proteinase K could disrupt associations between protein and RNA molecules in the fixed specimens that could ultimately lead to diffuse or weaker staining. Figure 2 shows that these few optimizations improved the Carnoy's fixations in terms of increased signal to noise ratios and cell specific staining. For example, Smed-slit and Smed-wnt-2 showed discernible staining, whereas they did not in the standard Carnoy's condition (Fig. 1, row 1). Similar to Figure 1, Figure 2 shows that FA had superior signal over background and well-defined cellular resolution. For example, smedbmp4-1 (Reddien et al., 2007), smedwi-1, Smed-slit, and Smed-wnt-2 hybridizations resolved sharply in much more detailed staining patterns in FA-fixed animals. For the H94.12e probe, delicate and unknown cells which stain in the dorsal epithelia (as well as the lack of midline staining) were not readily observed using Carnoy's.
Figure 2
Figure 2
FA-fixation is superior to optimized Carnoy's fixation
Finally, Carnoy's fixed animals consistently displayed lack of cellular transcript retention. When using the strong probe H1.3b, for example, we observed diffuse signal near robust expression domains (red arrows, Fig. 2). Such diffuse staining appeared real, but comparison with FA fixed animals (green arrows, Fig. 2) suggested instead that it likely represented transcript that had leeched out of expressing cells. In order to support this conclusion, we attempted to detect a ~22 nucleotide long microRNA (miRNA). We hypothesized that miRNAs should readily leech out of cells in Carnoy's fixed animals. Previous studies have identified numerous planarian miRNAs (Palakodeti et al., 2006), yet their expression patterns are unknown. We examined miR-124a because it represents a conserved miRNA that is expressed in the brain and nervous system in diverse organisms such as zebrafish, flies, and mice (Kloosterman and Plasterk, 2006; Kapsimali et al., 2007). The miRNA was detected using a short DNA-labeled locked nucleic acid oligonucleotide obtained commercially (Exiqon, probes hybed at 50°C, used at a 1:500 dilution). We observed strong staining for miR-124a in the brain of FA-fixed animals, but little to no staining was detected in Carnoy's-treated animals. This is the first example of whole-mount detection of a miRNA in planaria, and also shows that our optimized FA fixation achieves a fine balance of permeabilization and detection.
Optimized FA WISH protocol was used to detect anatomical structures at unprecedented resolution by colormetric and fluorescent development in planarians
To verify that our new high-resolution WISH methodology was suitable to detect specific planarian anatomical structures, we selected previously published genes with various anatomical expression patterns. On the basis of cell morphology in macerates, it has been estimated that planarians contain roughly 40 cell types (Sánchez Alvarado and Kang, 2005). However, using several markers expressed in unknown cell types, our data suggest that this may be a low estimate. These markers demonstrate a new level of cell and molecular complexity within the planarian body plan, and define specific anatomies via gene expression (Fig. 3). For example, unknown cell types were revealed at various levels along the anterior-posterior axis (Fig. 3A-D), and in structures associated with the pharynx, which is a major organ system in planarians (Fig. 3E-H). The excretory, gastrovascular, and nervous systems were also readily visualized with our optimized WISH protocol (Fig. 3I-K). Finally, this protocol is amenable to double fluorescent in situ hybridization (FISH) and to antibody staining using 3 different fluorophores, thus allowing the simultaneous visualization of multiple cell types and organ systems within the same animal (Fig. 3L). This result illustrates that our protocol does not overtly destroy antibody epitopes, even after the FISH detection of 2 separate riboprobes (Fig. 3L). Combined, these data show that the optimized protocol described here can be used to assay co-expression and/or multiple cell types using combinations of riboprobes with antibodies, all in whole-mount animals and at cellular resolution - a feat that has yet to become a reality for most other adult model organisms.
Figure 3
Figure 3
Fine anatomical structures detected at cellular resolution and by fluorescence
In a broader context, we have shown that WISH in planarians using a cross-linking based fixative is not only possible, but more robust, sensitive and consistent than current Carnoy's-based methods. The optimizations described here are likely to have widespread applicability to other systems, especially in animals that are aquatic and produce large amounts of mucus. For example, the use of NAC to chemically remove mucus provides avenues to explore gene expression in organisms not traditionally amenable to WISH analysis. Additionally, we found that the permeabilization steps using reduction and proteinase K do not appear to adversely affect many epitopes commonly detected with primary antibodies. The antibodies against alpha-tubulin and H3ser10p (which label pharynx/CNS and Histone H3 phosphorylated on Serine 10, respectively) are also easily detected after the WISH procedure (not shown). In addition, our data highlight that planarian stem cells must generate a wide array of differentiated cell types during both physiological turnover and regeneration of new tissue after injury. Essentially, any cell type that can be detected with a robust and sensitive WISH protocol can now be assayed in RNAi screens to find genes required for its patterning and regeneration. Furthermore, promoters from genes that label specific cells are potential candidates to drive tissue specific GFP in transgenic animals. Overall, our WISH optimizations are part of the common effort in the planarian community to advance the establishment of this organism as a powerful model system in biomedical research.
Animal Preparation
Note: Unless noted at particular steps, intact planarians may be nutated/rocked while early regenerating fragments (up to day 2) should be slowly rocked.
Day 1 (kill, remove mucus, fix, reduce/permeabilize, dehydrate, bleach)
  • 1) 
    Asexual planarians of a length between 2 and 4 mm were starved for one week and transferred either into 1.5ml Eppendorf-tubes (for processing up to 20 worms) or in 15ml Falcon tubes (for processing up to 200 worms).
  • 2) 
    Planarian water was replaced with 5% NAC solution, 5-10 minutes, room temperature (RT)
Notes:
  • * 
    1ml in 1.5ml Eppendorf tubes or 10ml in 15ml Falcon tubes
  • * 
    NAC is a mucolytic reagent that both kills the worms and removes their mucus
  • 3) 
    NAC was replaced with 4% Fixative, 15-20 minutes, RT
  • 4) 
    Fixative was removed and worms were rinsed 1X with PBSTx
  • 5) 
    PBSTx was replaced with preheated Reduction solution, 5-10 minutes, 37°C
Notes:
  • * 
    Reduction was carried out in a water bath with intermittent gentle agitation (specimens are fragile at this step)
  • * 
    Reduction aids with permeabilization to allow probe penetration
  • 6) 
    After removal of Reduction solution, worms were rinsed 1X with PBSTx
  • 7) 
    PBSTx was replaced with 50% Methanol solution, 5-10 minutes, RT
  • 8) 
    50% Methanol solution was replaced with 100% Methanol, 5-10 minutes, RT
Notes:
  • * 
    Specimens were then stored at -20°C for at least 1 hour (or up to several months)
  • * 
    Ethanol can be used in place of Methanol in steps 7-12, which seems better for long-term storage at -20°C, but may cause slightly weaker staining overall.
When ready (bleach)
  • 9) 
    Methanol was replaced with 6% Bleach solution, under direct light, overnight, RT
Note: This step removes pigment from the animal to help with visualization of the signal
  • 10) 
    6% Bleach solution was removed and specimens were rinsed twice with 100% Methanol
Note: After this, specimens were then returned to -20°C or used immediately
Day 2 (rehydrate, proteinase K, post-fix, hybridization)
  • 11) 
    100% Methanol was replaced with 50% Methanol solution, 5-10 minutes, RT
  • 12) 
    50% Methanol solution was replaced with PBSTx, 5-10 minutes, RT
  • 13) 
    PBSTx was replaced with Proteinase K solution, 10 minutes, RT
  • 14) 
    Proteinase K solution was replaced with 4% fixative, 10 minutes, RT
  • 15) 
    Fixative was removed and specimens were rinsed 2X with PBSTx
Hybridization
Note: When using an in situ robot (Intavis), specimens were added to robot for the hybridization steps
  • 16) 
    Wash in 1:1 (PBSTx:PreHyb), 10 minutes, RT
  • 17) 
    1:1 mix was replaced with Prehyb, 2hrs, 56°C
  • 18) 
    Prehyb was replaced with Riboprobe mix, >16hrs, 56°C
Note: Digoxigenin (DIG) labeled riboprobes were used for all specimens developed with NBT/BCIP
Day 3 (washing and antibody incubation)
Note: You do not need take precautions against RNAse-contamination after hybridization because RNAses from your hands and bacteria etc. are generally single-stranded RNAses and will not cleave dsRNA. In fact, some WISH protocols treat with RNAse post-hybridization to cleave unbound probe.
  • 19) 
    Riboprobe mix was removed and stored at -20°C and specimens were washed with the following times and preheated (to 56°C) solutions:
2 × 30min 1:1 [Wash hyb: (2×SSC + 0.1% Triton-X)]
2 × 30min 2×SSC + 0.1% Triton-X
2 × 30min 0.2×SSC + 0.1% Triton-X
  • 20) 
    Specimens were then allowed to return to RT and washed with MABT 2X 10min RT
Antibody incubation and development
Note: If using an in situ robot, specimens were transferred to 24 well plates, each step was carried out with nutating or rocking
  • 21) 
    Specimens were transferred to 24-well plastic plates, and solution replaced with Block solution, 1-2 hrs. at RT, or overnight at 4°C
  • 22) 
    Block solution was replaced with Antibody solution, 4hrs at RT, or overnight at 4°C
Day 4 (antibody washes and development)
  • 23) 
    Antibody solution was removed and specimens were rinsed with MABT
Note: Antibody solution may be recovered and used at least 2 more times over the course of a couple weeks
  • 24) 
    Specimens were then rinsed at least 6 more times with MABT, 20min each
  • 25) 
    MABT was replaced with AP buffer, 10min, RT
  • 26) 
    AP buffer was replaced with Development buffer and placed in the dark
  • 27) 
    Rate and extent of probe development was monitored under a dissection microscope and stopped once an optimal signal-to-background ratio was reached
Notes:
  • * 
    Time of development ranges from ~20min to >8hrs on a probe-by-probe basis
  • * 
    Development taking longer than 3-5hrs was continued overnight at 4°C
  • 28) 
    Development was stopped by replacing Development buffer with PBSTx
  • 29) 
    Specimens were post-fixed with 4% Fixative, 10min, RT
  • 30) 
    4% Fixative was removed and specimens were rinsed with PBSTx
  • 31) 
    PBSTx was replaced with 100 % Ethanol, ~20min, RT
Notes:
  • * 
    This step removes non-specific background staining
  • * 
    Specimens remained in ethanol until optimal signal to background ratio was reached and the NBT/BCIP precipitate had turned dark blue
  • 32) 
    100% Ethanol was replaced with 50% Ethanol Solution, 5min, RT
Note: Wait until specimens sink before continuing. This can be aided by adding drops of 50% Ethanol Solution directly on top of floating specimens.
  • 33) 
    50% Ethanol Solution was replaced with PBSTx
  • 34) 
    PBSTx was replaced by 80% Glycerol solution and stored at 4°C
  • 35) 
    Cleared specimens (i.e., no longer floating) were transferred to a slide and mounted under a #1-weight coverslip.
  • 36) 
    Slides were stored at 4°C until viewing or imaging on a Lumar dissecting scope (Zeiss), equipped with an Axiocam digital camera (Zeiss).
Animal preparation
5% NAC solution: 5% N-acetyl cysteine (NAC; Sigma) dissolved in 1X PBS.
Note: 5% NAC solution should be stored at 4°C and appears to be stable > 6 months, although the activity declines somewhat during this period.
PBSTx: 1X PBS + 0.1-0.5% Triton-X 100
4% Fixative: prepared fresh for each experiment by dilution of a 36.5 % formaldehyde stock solution (Formalin; Sigma) into PBSTx
Reduction solution: 50mM DTT, 1% NP-40, 0.5% SDS, in 1X PBS
50% Methanol solution: equal volumes of 1X PBSTx and 100 % Methanol
50% Ethanol solution: equal volumes of 1X PBSTx and 100 % Ethanol
6 % Bleach solution: 6% H2O2 (30% stock; Sigma) in Methanol
Proteinase K solution: 2μg/ml Proteinase-K (Invitrogen), 0.1% SDS, in 1X PBSTx
Hybridization
Hyb: 50-55% De-ionized Formamide (Roche)
5-10% Dextran Sulfate (Sigma, from 50% stock)
5x SSC
1mg/ml yeast torula RNA (Sigma)
1% Tween-20 (Sigma, from 10% stock)
Notes:
  • * 
    SDS may be used instead of Tween-20, but tends to precipitate during storage at -20°C.
  • * 
    Fresh de-ionization of formamide appears to affect staining differently in different organisms, but is critical for planarians. We de-ionize 1L of formamide (Roche) with 50g of Bio-RadAG 501-X8 (D) Resin for 1hr at RT, then filter, aliquot, and store at -80°C.
Prehyb / Wash Hyb: Hyb without the dextran sulfate
Riboprobe mix: 400μl Hyb plus ~400ng (~4μl) of riboprobe
Note: Riboprobe mix was denatured at 72-90°C for 5 minutes, then placed at 56°C prior to use
MABT: 100mM maleic acid, 150mM NaCl, 0.1% Tween-20, pH to 7.5 with NaOH
SSC: 20X SSC stock (Sigma)
Antibody incubation and development
Blocking solution: 5-10% horse serum in MABT
Note: Head-to-head comparisons indicate that bovine serum albumin (BSA) in the blocking solution is detrimental to signal detection
Antibody solution: Antibody diluted into Blocking solution
Note: anti-DIG-AP (Roche) was used at 1:4000 for all NBT/BCIP experiments
AP buffer: 100mM Tris, pH 9.5; 100mM NaCl; 50mM MgCl2; 0.1 % Tween-20 brought up to volume with 10% polyvinylalcohol solution (PVA; Sigma P8136). This was prepared fresh prior to every experiment from stocks of 1M Tris, pH 9.5; 5M NaCl; 1M MgCl2; 10% tween-20. The PVA solution is a 10% w/v stock in H2O stored at RT.
Development buffer: Freshly made AP buffer with 4.5μl/ml NBT (Roche) and 3.5μl/ml BCIP (Roche)
Note: PVA greatly increases AP activity and helps the most for weak probes. 80% Glycerol solution: 80% Glycerol; 10mM Tris, pH 7.5; 1mM EDTA
Antisense-riboprobes were synthesized from PCR-templates using either T7 or T3 RNA-polymerase (Promega). Probe synthesis was carried out for 2 h at 37°C using digoxigenin (DIG (Tautz and Pfeifle, 1989)), fluorescein (Hauptmann and Gerster, 1996), biotin (Hauptmann and Gerster, 1996), or dinitrophenol (DNP (Denkers et al., 2004)), -labeling mix and 400ng of DNA template. DNA was then degraded with DNAse (Promega) for 15min at 37°C. Probes were hydrolyzed by adding 60μl of Carbonate Buffer (80mM Na2CO3, 53.3mM NaHCO3; pH 10.2) and incubating at 65°C for 2-3min. The hydrolysis-reaction was quenched with 80μl of Stop solution (200 mM NaAc, pH to 6.0; adjusted with Acetic Acid). Hydrolyzed probes were precipitated by adding 40μg Glycogen (20mg/ml stock solution, Roche) and 400μl ice-cold 100% ethanol, and then centrifuged at high speed at 4°C for 40 minutes. The resulting pellet was dried briefly after complete aspiration of the supernatant and dissolved in 100μl of Hyb solution. Riboprobe stocks were stored at -80°C. For use, riboprobes stocks were generally diluted 1:100 or 1:200 into 400-500μl of Hyb solution.
Animal preparation and hybridization was carried out as detailed above in the appropriate sections. The protocol only differs during the Antibody incubation and development steps and was adapted from the protocols on the weblinks below. Sequential rounds of Tyramide signal amplification were used for fluorescent probe development. FITC-Tyramide and Cy3-Tyramide were synthesized according to the protocols of Lance Davidson and Peter Vize, respectively (FITC-Tyramide: http://www.engr.pitt.edu/ldavidson/fluor_insitu/fluorescent_in_situ.html Cy3-Tyramide: http://www.xenbase.org/other/static/methods/FISH/Cy3_tyramide.jsp). Riboprobe-antibodies included anti-DIG-POD (1:500, Roche), anti-fluorescein-POD (1:300, Roche), streptavidin-POD (Roche), or anti-DNP-HRP (1:100, Perkin-Elmer). After the initial riboprobe-antibody incubation, specimens were washed 6x for 20min in MABT and subsequently 1× 30min in PBSTI (PBSTx + 10mM Imidazole). Specimens were pre-incubated for 30 minutes in 400μl of the appropriate Tyramide dilution (FITC-Tyramide: ~1:1000; Cy3-Tyramide: ~1:500; dilutions in PBSTI). Development was initiated by adding H2O2 to a final concentration of 0.002-0.015 % (H2O2 should be freshly diluted in PBSTI from a 30% stock) and allowed to proceed for 45 minutes under constant agitation on a shaker in the dark. Specimens were rinsed 2x 5 minutes in PBSTx, remaining peroxidase-activity was quenched by incubating for 45 minutes in 1% H2O2 in PBSTx, followed by 4 rinses with MABT. Incubation with the second riboprobe-antibody (directed against the second riboprobe) and subsequent detection were carried out exactly as above, except that the blocking step was omitted.
Non-riboprobe antibody staining was performed during the riboprobe-antibody steps above. Specifically, after incubating specimens with the first riboprobe-antibody (anti-DIG-POD) for 4 hours at RT, washes were performed with Blocking solution. Specimens were incubated with VC-1 antibody (diluted 1:10,000 in Antibody solution) overnight at RT. Following washes with MABT, the first riboprobe was developed and the reaction quenched. After incubation with the second riboprobe-antibody for 4hr at RT, specimens were washed in Blocking solution and then incubated overnight at RT with secondary antibody (anti-mouse-Alexa Fluor 647) for VC-1. After the appropriate washes, the second riboprobe was developed with tyramide signal amplification. Following at least 6× 20 minutes washes with MABT, specimens were mounted in 80 % Glycerol solution under #1-weight coverslips and imaged on a Zeiss 510 Live confocal microscope.
Acknowledgments
This work was funded in part by NIH-NIGMS RO-1 GM57260. BJP was supported by Damon Runyon Foundation Grant #1888-05; GTE by NIH Developmental Biology Training Grant 5T32 HD07491; KAG by NIH-NIGMS F32GM082016; JCR by the European Molecular Biology Organization. ASA is a Howard Hughes Medical Institute investigator.
  • Cebria F, Guo T, Jopek J, Newmark PA. Regeneration and maintenance of the planarian midline is regulated by a slit orthologue. Dev Biol. 2007;307:394–406. [PMC free article] [PubMed]
  • Denkers N, Garcia-Villalba P, Rodesch CK, Nielson KR, Mauch TJ. FISHing for chick genes: Triple-label whole-mount fluorescence in situ hybridization detects simultaneous and overlapping gene expression in avian embryos. Dev Dyn. 2004;229:651–657. [PubMed]
  • Eisenhoffer GT, Kang H, Sánchez Alvarado A. Molecular analysis of stem cells and their descendants during cell turnover and regeneration in the planarian Schmidtea mediterranea. Cell Stem Cell. 2008;3:327–339. [PMC free article] [PubMed]
  • Gurley KA, Rink JC, Sánchez Alvarado A. Beta-catenin defines head versus tail identity during planarian regeneration and homeostasis. Science. 2008;319:323–327. [PMC free article] [PubMed]
  • Hauptmann G, Gerster T. Multicolour whole-mount in situ hybridization to Drosophila embryos. Development Genes & Evolution. 1996;206:292–295. [PubMed]
  • Hyman LH. The Invertebrates: Platyhelminthes and Rhynchocoela. McGraw-Hill Book Company, Inc.; 1951.
  • Kapsimali M, Kloosterman WP, de Bruijn E, Rosa F, Plasterk RH, Wilson SW. MicroRNAs show a wide diversity of expression profiles in the developing and mature central nervous system. Genome Biol. 2007;8:R173. [PMC free article] [PubMed]
  • Kloosterman WP, Plasterk RH. The diverse functions of microRNAs in animal development and disease. Dev Cell. 2006;11:441–450. [PubMed]
  • Morgan TH. Experimental studies of the regeneration of Planaria maculata. Arch. Entw. Mech. Org. 1898;7:364–397.
  • Newmark PA, Sánchez Alvarado A. Not your father's planarian: a classic model enters the era of functional genomics. Nat Rev Genet. 2002;3:210–219. [PubMed]
  • Palakodeti D, Smielewska M, Graveley BR. MicroRNAs from the Planarian Schmidtea mediterranea: a model system for stem cell biology. Rna. 2006;12:1640–1649. [PubMed]
  • Petersen CP, Reddien PW. Smed-betacatenin-1 is required for anteroposterior blastema polarity in planarian regeneration. Science. 2008;319:327–330. [PubMed]
  • Reddien PW, Bermange AL, Kicza AM, Sánchez Alvarado A. BMP signaling regulates the dorsal planarian midline and is needed for asymmetric regeneration. Development. 2007;134:4043–4051. [PubMed]
  • Reddien PW, Oviedo NJ, Jennings JR, Jenkin JC, Sánchez Alvarado A. SMEDWI-2 is a PIWI-like protein that regulates planarian stem cells. Science. 2005;310:1327–1330. [PubMed]
  • Sánchez Alvarado A, Kang H. Multicellularity, stem cells, and the neoblasts of the planarian Schmidtea mediterranea. Exp Cell Res. 2005;306:299–308. [PubMed]
  • Tautz D, Pfeifle C. A non-radioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals translational control of the segmentation gene hunchback. Chromosoma. 1989;98:81–85. [PubMed]
  • Umesono Y, Watanabe K, Agata K. A planarian orthopedia homolog is specifically expressed in the branch region of both the mature and regenerating brain. Dev Growth Differ. 1997;39:723–727. [PubMed]
  • Urieli-Shoval S, Meek RL, Hanson RH, Ferguson M, Gordon D, Benditt EP. Preservation of RNA for in situ hybridization: Carnoy's versus formaldehyde fixation. J Histochem Cytochem. 1992;40:1879–1885. [PubMed]