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Histological techniques are critical for observing tissue and cellular morphology. Here, we outline our protocol for embedding, serial sectioning, staining, and visualizing zebrafish embryos embedded in JB-4™ plastic resin – a glycol methacrylate-based medium that results in excellent preservation of tissue morphology. In addition we describe our procedures for staining plastic sections with Toluidine Blue or Hematoxylin and Eosin (H&E), and show how to couple these stains with whole-mount RNA in situ hybridization. We also describe how to maintain and visualize immunofluorescence and GFP signals in JB-4™ resin. The protocol we outline – from embryo preparation, embedding, sectioning and staining to visualization - can be accomplished in three days. Overall, we reinforce that plastic embedding can provide higher resolution of cellular details and is a valuable tool for cellular and morphological studies in zebrafish.
Histology is a classic technique that is used to study the morphology of cellular and sub-cellular structures by cutting specimens into thin sections. Even with real-time imaging technology, histological sectioning and staining remain necessary techniques to visualize cellular morphology in tissues. Histotechniques are important tools for understanding the progression of tissue development and addressing phenotypes related to mutant backgrounds or pathological states.
Zebrafish are an attractive model for studying tissue morphogenesis, owing in part to their optical clarity during embryonic stages. Sectioning through the entire embryo is feasible because embryos are small at embryonic and larval stages (around 1mm)1. Furthermore, bones have not matured at embryonic stages2, so the tissue remains soft and provides less resistance when sectioning. Here, we present our optimized protocols for serial sectioning zebrafish embryos with JB-4™ plastic resin. Our protocol for embedding and sectioning zebrafish embryos in JB-4™ resin (Figure 1) has been adapted from the protocol originally provided for JB-4™ by Electron Microscopy Sciences and optimized for zebrafish embryos. Work from our laboratory has used these protocols to analyze kidney and heart development in zebrafish3–8. We also provide information on how to couple JB-4™ embedding with whole-mount RNA in situ hybridization, immunofluorescence and GFP fluorescence. Our protocol can be accomplished in three days as outlined in Figure 1.
Depending on the type of analysis necessary for a given experiment, there are many histological techniques available, each having their own advantages and disadvantages. Among the most common techniques are frozen/cryoembedding, paraffin embedding, and plastic embedding for sectioning. For examples utilizing zebrafish embryos please see9–11. Cryoembedded samples do not need to be fixed prior to embedding so sections can be prepared quickly and epitopes are typically well preserved in the sample; however, the cellular morphology is generally poor due to the freezing process. Samples used for paraffin embedding require fixation before the embedding procedure and more tissue processing steps than cryoembedding; however, morphology is greatly improved. In both systems, immunofluorescence and RNA in situ hybridization can be performed after sectioning.
JB-4™ is a glycol methacrylate-based polymer used as embedding material that can be used to cut semi-thin or thicker sections. The main advantage to embedding in plastic resins, such as JB-4™, is the ability to easily produce ultra-thin (0.5–1uM) or semi-thin (2–3um) sections depending on the plastic embedding medium12. It has long been appreciated that thin sections provide an increased level of cytological detail over thicker sections. While embedding and sectioning in paraffin can provide reasonably good results, semi-thin sections are difficult to produce in paraffin, and the artifacts produced in wax can limit any improvement in cellular resolution that thinner sectioning would provide12. Thus we recommend our protocol for any analysis where preservation of cellular detail is critical. In addition, processing embryos for paraffin embedding takes many more steps and requires dehydration and clearing agents such as xylene which are often toxic and difficult to dispose of. While paraffin embedding can be done without special equipment, it is typically performed using processing machines which minimize exposure to toxic clearing agents, but may not be available to some users, or may process samples inefficiently. By contrast, plastic embedding requires no special processing equipment, can be performed with or without dehydration, and samples can be stored after embedding indefinitely if processing for morphology. Our protocol is also recommended when sectioning embryos that have previously been processed for RNA in situ hybridization or immunofluorescence, both of which can be altered by the multiple steps involved in paraffin processing.
While JB-4™ resin has some distinct advantageous in histology, RNA in situ hybridization and immunofluorescence must be performed in wholemount, prior to embedding and sectioning samples. Thus, our protocol is not recommended for analyses where these procedures would be more informative if performed following sectioning. Sample orientation in JB-4™ resin can also be challenging because plastic resins are initially water-like, unlike the viscous mediums in frozen and paraffin embedding. But, as we describe in this paper any desired orientation can be achieved utilizing multiple embedding steps.
Using the protocol described in this paper, we have been successful in embedding and sectioning zebrafish embryos at multiple stages; from as early as 30% epiboly to adults. This protocol is recommended for any analysis where preservation of cellular detail is critical, or where sectioning of embryos is desired following wholemount RNA in situ hybridization or immunofluorescence. When performing sectioning following RNA in situ hybridization, best results are achieved with probes that provide strong signals since the amount of signal per section will be a fraction of the total stain. While embryos being processed for histology can be stored indefinitely once embedded, embryos that are processed for RNA in situ hybridization or immunofluorescence should be sectioned and analyzed immediately after embedding to prevent a loss or reduction of signals. When analyzing mutant or morpholino injected embryos, it is important to process wildtype/control embryos in parallel to have samples for comparison to determine if any potential phenotypes are real, or an artifact of processing.
Purchase as a powder (Sigma) or liquid (Electron Microscopy Sciences). If purchased as powder, dissolve 4g of PFA in 100mls of PBS, dissolve at 68, cool to room temperature (20–23°C) and adjust pH to 7.4. Paraformaldehyde solution can be purchased in different concentrations. We typically purchase 20% solutions and dilute to 4% in PBS. Store in aliquots at −20°C for up to 18 months. CAUTION! Work in a well-ventilated space and wear protective eye wear and gloves when handling PFA.
Make according to the manufacturer's protocol. Add 0.625g of Benzoyl Peroxide (catalyst—provided with kit) to 50ml of JB-4™ Solution A (provided in kit). We make this solution in a 50ml conical tube and rock at room temperature for 20 min. or until dissolved. Store at 4°C in the dark for one month. CAUTION! Work in a well-ventilated space and wear protective eye wear and gloves when handling Benzoyl Peroxide.
Make according to the manufacturer's protocol. Add 1ml of JB-4™ Solution B (accelerator-provided with kit) to 25ml of Infiltration Solution (prepared as described above). Make this solution in a 50ml conical tube, and mix by using a plastic pipette. This solution will begin to solidify quickly, so immediately add to the embedding molds. After adding it to all the molds, place the cap back on the conical tube and allow the remainder to polymerize. This will serve as a test to determine if the polymerization reaction was successful. CAUTION! Work in a chemical hood and wear protective eye wear and gloves when handling all liquid components of JB-4™ resin.
CRITICAL! Staining solutions cannot contain alcohol! Alcohol will cause the plastic sections to lose adherence to the glass slide.
Gill's #2 Hematoxylin – can purchase solution or make your own. Store at room temperature for up to 2 years.
|1 Hematoxylin (0.4% weight/volume)||4g|
|2 Sodium Iodate (0.04% weight/volume)||0.4g|
|3 Aluminum Sulfate (3.5% weight/volume)||35g|
|4 Ethylene glycol (25%)||250ml|
|5 Acetic Acid (4%)||40ml|
|6 dH20 to desired final amount||bring to 1L|
Eosin-Y – Store at room temperature for up to 1 year.
|1 Eosin-Y (0.5% weight/volume)||5g|
|2 dH20 to desired final amount||bring to 1L|
Scott's Tap Water Substitute – can purchase solution or make your own. Store at room temperature for up to 1 year.
|1 Sodium Bicarbonate (NaHCO3)||2g|
|2 Magnesium Sulfate Heptahydrate (MgSO4·7H2O)||20g|
|3 dH20 to desired final amount||bring to 1L|
Scott's Tap Water Substitute is a “bluing” reagent at the correct pH to enhance the blue color of stained nuclei.
Toluidine blue solution – Store at room temperature for up to 18 months
|1 Toluidine Blue O (1% weight/volume)||10g|
|2 dH20 – to desired final amount||bring to 1L|
TIMING Variable (typically overnight at 4°C)
TIMING 1–2 hours, then overnight
TIMING ~30–45 min
Toluidine blue is a metachromatic dye that stains cellular components such as the nuclei and cytoplasm various shades of blue and tissues such as the cartilage a pinkish color (probably due to its high proteoglycan content14). Toluidine blue staining procedures are quick, and only require a 1% Toluidine blue solution.
The purpose of a Haematoxylin and Eosin (H&E) stain is to broadly distinguish nuclear material from cytoplasmic material15.
The Haematoxylin is a basic dye with a high affinity towards basophilic structures (such as nucleic acids, which are concentrated in the nucleus) and yields a dark purple stain. The Eosin is an acidic dye with a high affinity towards eosinophilic structures (such as the protein rich cytoplasm) and yields a pink stain15. The dark purple nuclei have better contrast with the pink cytolplasmic stain than the toluidine blue stain; however the cartilage is not as easily identifiable.
Any methods involving fixation, processing and sectioning can result in shrinkage or swelling of the sample. Improper sectioning can further stretch and distort tissues. However we find that embryos processed correctly with this protocol show little distortion as measured by notochord cell diameter. These cells are highly vacuolated when mature, and can serve as a strong indicator of shrinkage in a tissue. The diameter of the mature notochord is ~50μm16. The notochord diameter was measured in two different images using ImageJ software and found to be 56 and 59μm which may indicate a minimal amount of swelling.
RNA in situ hybridization is typically performed by standard methods in whole-mount zebrafish embryos13. However, it is often difficult is determine precise tissue localization of the RNA expression. Since staining from either 3,3'-Diaminobenzidine (DAB) or BCIP/NBT substrate applications is maintained in JB-4™ resin, histological analysis can drastically improve the cellular resolution of RNA expression. It is often desirable to take a picture of the sections without an H&E stain, as this stain will obscure weaker DAB or BCIP/NBT stains. However, when the in situ stain is strong (as in Figure 4C), the RNA expression domains are clearly distinguished from the H&E stain.
In analyzing protein expression, determining cellular localization is often desired in addition to tissue localization. Although confocal microscopy can provide optical sectioning through samples, zebrafish embryos are often too thick for good resolution deep into the tissue in whole-mount, and morphology cannot be appropriately analyzed. Therefore, sectioning is often critical to examine cellular localization of proteins. An example of a section through an immunostained embryo in whole mount is pictured in Figure 4D. JB-4™ resin can also be used to analyze GFP signals in sections – from both mosaically expressing (Figure 4E) and transgenic (Figure 4F) fish.
JB-4™ resin-based histology beautifully maintains morphology, enabling a close examination of cellular phenotypes, RNA expression, and protein localization. Although there are some challenges to using JB-4™ resin, the high level of tissue preservation and cellular resolution make it ideal for analysis of zebrafish morphology.
TIMING ~1–3 hours
One of the most critical steps in histology is obtaining proper orientation of the sample. The JB-4™ resin embedding solution is initially water-like, thus positioning embryos is difficult compared to using more viscous embedding solutions such as those used in cryoembedding. Although it is possible to embed a large number of samples and use the embryos that by chance are in the best orientation, we prefer to use multiple embedding steps to get a consistent and desired orientation and waste fewer samples.
Sectioning TIMING ~ 1–1.5hours/sample
Staining procedures are performed many different ways. In our lab, we use glass staining dishes that will hold the dye and a glass slide rack that can be dipped into the dishes and transferred to new dishes with the appropriate solution. These are described in the Equipment section of this paper.
CAUTION! Wear protective gloves and lab coat.
A summary of the approximate time needed to complete each stage of the procedure is provided in a flow diagram presented in Figure 1.
Troubleshooting advice is provided in Table 1.
We thank Kari Baker, Kim Jaffe, Noriko Okabe, Jon Rosen, and Jodi Schottenfeld for providing samples and help with the protocol; Michael Pack for advice on immunofluorescence protocols; Valantou Grover for information on storage conditions for staining reagents; and members of the Burdine lab for critical reading of the manuscript. R.D.B. is the 44th Scholar of the Edward Mallinckrodt Jr. Foundation, and funds from this award were used in support of this work. Funds from awards to R.D.B. from the New Jersey Commission on Cancer Research (04-2405-CCR-E0) and from the Polycystic Kidney Disease Foundation, (#117b2r) were used in support of this work. J.S.B. was supported by predoctoral award 05-2411-CCR-E0 from the New Jersey Commission on Cancer Research.
Competing financial interests The authors declare that they have no competing financial interests.