Optimization of Whole-Body Zebrafish Sectioning Methods for Mass Spectrometry Imaging

Mass spectrometry imaging (MSI) methods and protocols have become widely adapted to a variety of tissues and species. However, the MSI literature contains minimal information on whole-body cryosection preparation for the zebrafish (ZF; Danio rerio), a model organism routinely used in developmental, toxicity, and carcinogenicity studies. The optimal medium for embedding and cryosectioning a whole organism or soft-tissue specimen for histological examination is a synthetic polymer mixture that is incompatible with MSI as a result of ion suppression. We describe the optimal methods and results for embedding and cryosectioning whole-body ZF for MALDI-MSI. We evaluated 13 distinct embedding media formulations and found a supportive hydrogel with the consistency of cartilage to be the optimal embedding medium. The hydrogel medium does not interfere with MSI data collection, aids in tissue stability, is readily available for purchase, and is easy to prepare and handle during cryosectioning. Additionally, we decreased the matrix cluster interference commonly caused by α-cyano-4-hydroxycinnamic acid by adding ammonium phosphate to the solvent spray solution. The optimized methods developed in our laboratory produced high-quality cryosections, as well as high-quality mass spectral images of sectioned ZF.     

Developmentally regulated expression of a mitogen-activated protein kinase in Xenopus laevis.

Mitogen-activated protein (MAP) kinases are activated in somatic cells in response to many extracellular stimuli and in oocytes during meiotic maturation. We have examined the tissue specificity of expression of a MAP kinase (Xp42) in adult and larval Xenopus laevis. MAP kinase RNA and protein were abundant in the nervous system and lymphoid tissues and were readily detected in most other organs. A remarkably high level of RNA was detected in ovary. Fractionation of oocytes showed that MAP kinase RNA is expressed at the highest level in small oocytes, suggesting that it is a maternal RNA that is stored for early embryogenesis. The levels of MAP kinase RNA and protein did not change from the time of fertilization through to late blastula. The results are consistent with functions for MAP kinases in signal transduction in embryonic as well as adult cells.     

Enolase isoenzymes in adult and developing Xenopus laevis and characterization of a cloned enolase sequence.

As part of a study of glycolysis during early development we have examined the pattern of expression of enolase isoenzymes in Xenopus laevis. In addition, the nucleotide sequence of a cDNA clone coding for the complete amino acid sequence of one enolase gene (ENO1) in X. laevis was determined. X. laevis ENO1 shows highest homology to mammalian non-neuronal enolase. Analysis of enolase isoenzymes in X. laevis by non-denaturing electrophoresis on cellulose acetate strips revealed five isoenzymes. One form was present in all tissues tested, two additional forms were expressed in oocytes, embryos, adult liver and adult brain, and two further forms were restricted to larval and adult muscle. Since enolase is a dimer, three different monomers (gene products) could account for the observed number of isoenzymes. This pattern of enolase isoenzyme expression in X. laevis differs from that of birds and mammals. In birds and mammals the most acidic form is neuron-specific and there is only one major isoenzyme expressed in the liver. RNAase protection experiments showed the presence of ENO1 mRNA in oocytes, liver and muscle, suggesting that it codes for a non-tissue-restricted isoenzyme. ENO1 mRNA concentrations are high in early oocytes, decrease during oogenesis and decrease further after fertilization. Enolase protein, however, is maintained at high concentrations throughout this period.     

The identification of a tissue-restricted plasma membrane marker in Xenopus laevis embryos by using a monoclonal antibody

A library of monoclonal antibodies raised against partially purified membrane fractions from Xenopus laevis oocytes has been produced. One of these antibodies has been cloned and characterized in detail. It was found to be specific for a membrane-bound antigen of apparent Mr, 55,000. The distribution of the antigen has been studied by indirect immunofluorescence on sections of X. laevis embryos and has been found to be highly specific for some ectodermal and endodermal tissues. It was not present on mesodermal tissues.     

Immunofluorescence localisation of microtubules in plant root tips embedded in butyl-methyl methacrylate

Most immunofluorescence studies of microtubules in plant cells have used enzymatically isolated cells whose position in the organ and stage of development was unknown. Moreover, attempts to label microtubules in cells in intact tissue have suffered from poor resolution and inferior tissue preservation. To overcome these difficulties, I have used a removable embedding resin to localise microtubules in situ. Tissues were fixed in 4% paraformaldehyde and 0.2% glutaraldehyde and embedded in butyl-methyl methacrylate. Semi-thin sections were extracted with acetone to remove the resin and labelled with anti-tubulin followed FITC-labelled second antibody. Brightly stained microtubule arrays were clearly visualized. This reversible embedding medium should provide a powerful tool to study developmental changes in microtubule arrays in growing and differentiating plant tissues.     

FURTHER STUDY RE-EXAMINING WHETHER LARVAL EPIDERMIS CAN OMIT THE SECRETION OF A PUPAL CUTICLE, USING THE SILKWORM, BOMBYX MORI

When larval tissue is exposed to a hormonal milieu lacking juvenile hormone, adult characters appear directly, omitting the pupal stage, in some insects but not in others, including Bombyx mori. An attempt was made to induce omission of pupal characters in this species by varying the stage of the larval epidermis to be tested. Pieces of larval integument taken from fourth- and fifth-instar larvae of various stages were transplanted to developing adults. Although the number of cuticle layers and the types of cuticle produced differed depending on the age of the donors, none of the pieces omitted secreting the pupal cuticle. It is concluded that the larval epidermis cannot omit secreting pupal cuticle, and that a transition of tissue competence may play an important part in the sequential appearance of larval, pupal, and adult characters.     

Piccolyte investments for better section cutting.

Piccolyte 115 (beta-pinence polymers) added to Tissuemat, Paraplast or Peel-Away embedding media is recommended for investment of infiltrated tissues. Mixed with paraffin at 3% and 10% and used for double embedding of paraffin infiltrated tissues, Piccolyte 115 permits good, complete sections virtually free of folds or wrinkles in less time and with less effort than with paraffin embedding alone.     

Metamorphic changes of wild type and mutant Drosophila tissues induced by 20-hydroxy ecdysone in vitro

Wild type (Oregon R) and non-pupariating as well as late-pupariating mutant larval tissues were cultured in vitro up to 5 weeks with and without 20-hydroxy ecdysone (1 μg/ml). The following responses were elicited by the hormone: in the case of wild type tissues detachment of the larval epidermis and muscles from the cuticle; puparial tanning and sclerotization of the larval cuticle; dissociation of the fat body into single cells; inhibition of the movement of the hind intestine. Most of these responses developed within 1 week of culturing. Of the 4 mutants tested, 3 behaved like the wild type. In cultures of ?(1)npr-1, however, puparial tanning, disc evagination, and inhibition of the movement of the hind intestine was abnormally weak and the dissociation of fat body was not observed at all. Detachment of the epidermis and muscles as well as formation of the pupal cuticle by disc tissue occurred normally. The results are discussed with respect to the ecdysteroid-induced metamorphosis of the tissues and the autonomy of mutant gene action.     

Piccolyte Investments for Better Section Cutting

Piccolyte 115 (β-pinene polymers) added to Tissuemat, Paraplast or Peel-Away embedding media is recommended for investment of paraffin infiltrated tissues. Mixed with paraffin at 3% and 10% and used for double embedding of paraffin infiltrated tissues, Piccolyte 115 permits good, complete sections virtually free of folds or wrinkles in less time and with less effort than with paraffin embedding alone.     

Immunocytochemical localization of human growth hormone- and prolactin-like antigenic determinants in the insects, Locusta migratoria and Sarcophaga bullata.

1. By use of the peroxidase-antiperoxidase immunocytochemical method, substances immunoreactive to antisera directed against human growth hormone (hGH) and prolactin (hPrl) were localized in the nervous system of larval and adult Locusta migratoria and of adult Sarcophaga bullata belonging to different age groups. 2. No major differences in the distribution of cerebral immunoreactive materials were observed between males and females or between juvenile and adult insects. 3. Differential immuno-labeling of alternating tissue sections demonstrated that materials resembling hGH or hPrl are present in distinct neurons in the locust, whereas neurons immunoreactive to both antisera were detected in the fleshfly (Sarcophaga).     

Expression of phenolic and tyrosyl ring iodothyronine deiodinases in Xenopus laevis oocytes is dependent on the tissue source of injected poly(A)+ RNA

The phenolic (5' position) and tyrosyl (5 position) ring deiodinases which catalyze the peripheral metabolism of thyroid hormones have proven difficult to purify and characterize biochemically. The present studies used Xenopus laevis oocytes as an in vivo translational assay system for detecting and quantitating mRNA for these enzymes. The injection of poly(A)+ RNA prepared from a human term placenta induced 5-deiodinase activity in oocytes. The expressed activity increased for up to 96 h after injection, was proportional to the amount of RNA injected, and manifested a Michaelis-Menten constant (Km) for T3 of 1.6 nM. In oocytes injected with poly(A)+ RNA prepared from rat liver, anterior pituitary gland, or brown adipose tissue, 5-deiodinase activity could not be demonstrated. The injection of poly(A)+ RNA from 15-day-old chick embryonic liver induced both 5'- and 5-deiodinase activity, with the 5'-deiodinase activity being sensitive to inhibition by 6-n-propyl-2-thiouracil. X. laevis oocytes can thus be induced to express either phenolic or tyrosyl ring deiodinase activity, or both, by the microinjection of poly(A)+ RNA prepared from selected tissues. These findings demonstrate that the types of deiodinase activity present in different organs represent tissue specific patterns of mRNA expression and strongly suggest that the enzymes responsible for types I and III deiodinase activity are encoded by different mRNAs.     

A modified anti-roll plate as a remedy for the ill-effects of electrical charge during cryosectioning

This report describes problems commonly encountered in cryosectioning of fragile tissues, such as jamming and scrambling of sections between knife and anti-roll plate. The electrostatic nature of the factors causing these derangements is demonstrated here using simple methods, and several modifications to the anti-roll plate surface are tested for their help in obviating these difficulties. The anti-roll plate material found to be most effective consists of a metal oxide-coated glass which is commercially available at low cost.     

Immunocytochemical localization of protein antigens in large sections of tissues embedded in water-soluble embedding media

JB4 and Immunobed are water-soluble embedding media used for embedding large blocks of tissue. Immunobed was specifically designed for immunocytochemistry because ethanol extraction of an additive in the monomer of the resin is reported to render tissue sections permeable to immunoglobulins. We have modified the manufacturer's protocol to accomplish localization of two protein antigens in tissues embedded in either JB4 or Immunobed. Luteinizing hormone-beta (LH beta) was localized in sections of rat and bovine pituitary tissues and bovine placental lactogen (bPL) was localized in sections of placentomes from bovine placentas. Sections received one of the following pre-treatments: absolute EtOH; NaHCO3 buffer, pH 6-10; EtOH followed by NaHCO3 buffer; one of several enzymes; EtOH followed by enzyme; NaHCO3 buffer followed by enzyme. Anti-LH beta stained only pituitary gonadotrophs and anti-bPL stained only placental binucleate cells, as assessed by absorption controls. Pre-treatment with enzyme was required for staining of sections, but an alkaline pH change (NaHCO3) had little or no effect. Ethanol pretreatment had little or no effect alone or in conjunction with NaHCO3 or enzyme. Sections were sufficiently thin (1.5 micron) to afford resolution of structure, but suitably large (approximately 2 cm2) to minimize problems of sampling.     

Embedding Plant Tissue with Plastic Using High Pressure: A New Method for Light and Electron Microscopy

An embedding technique has been developed to overcome difficulties that confront light and electron microscopists working with so-called “hard-to-embed” plant tissue. The method was originally described for freeze-dried material. It uses a modified Quickfit Rotaflo Valve and low heat to generate high pressure to aid in the infiltration and embedding of tissue with propylene oxide and plastic. The technique is not too cumbersome and requires 6 days from the dehydration step to the end of the polymerization process. Thick sections (1-2 μm) obtained from material prepared by this method stain readily with toluidine blue, and thin sections for the electron microscope stain satisfactorily following standard treatment with uranyl acetate and lead citrate. The thin sections are stable under the beam of the electron microscope. Results indicate that the quality of tissue preservation with this high pressure embedding technique is as good as that observed using standard embedding methods for electron microscopy.     

Bufo marinus oocytes as a model for ion channel protein expression and functional characterization for electrophysiological studies.

Oocytes from Xenopus laevis are commonly used as an expression system for ion channel proteins. The aim of this study was to determine whether oocytes from the Colombian native toad, Bufo marinus, could be used as an alternative expression system for ion channel protein expression and functional characterization using the two-microelectrode voltage clamp method. B. marinus oocytes and X. laevis were isolated and cultured in similar conditions. The mean resting membrane potential of B. marinus oocytes was similar to that of X. laevis oocytes as well as the whole-cell basal currents. The potassium ion channel Kv1.1 was successfully expressed in B. marinus oocytes and showed a typical outward rectifying current. Potassium channel blockers reduced these currents. The similarities on electrical properties and expression of ion channel proteins show that B. marinus oocytes can be used effectively to express these proteins, making these cells a viable heterologous system for the expression of ion channel proteins and their electrophysiological characterization.     

Xanthine dehydrogenase activity in the clawed frog, Xenopus laevis

Xanthine dehydrogenase (XDH; EC 1.2.1.37) activity in the clawed frog, Xenopus laevis, was detected in kidney tissue homogenates, but not in skin, liver, ovaries or gut tissues. The enzyme migrated as a single band of activity on both polyacrylamide and starch gel electropherograms, exhibited substrate inhibition, and did not appear developmentally until feeding larval stages. The tissue specificity, post-fertilization stage of appearance and single isozymic form make this a useful enzyme marker for further study concerning its developmental appearance and maintenance as a kidney-specific protein.     

Evaluation of ethanol-fixed,paraffin-embedded tissues for proteomic applications

We previously reported that ethanol fixation and paraffin embedding of tissues produce excellent histomorphology and good preservation of macromolecules. Here, we present a detailed evaluation of ethanol-fixed tissues for proteomic initiatives. When proteins were extracted from ethanol-fixed, paraffin-embedded prostate tissue, resolved by two-dimensional gel electrophoresis (2-DE), and stained by standard methods, several hundred protein molecules could be detected and successfully analyzed by mass spectrometry. Protein profiles obtained from ethanol-fixed tissues were highly similar to those observed from frozen tissues, in contrast to the poor protein recovery from formalin-fixed material. The protein content of specific cells that were microdissected from ethanol-fixed tissue sections using laser capture microdissection could also be successfully analyzed by 2-DE. We observed that eosin staining of tissue sections had a detrimental effect on protein separation, whereas hematoxylin staining had minimal consequence. In order to illustrate the applicability of ethanol-fixed tissues for proteomic discovery studies, we compared the protein profiles of patient-matched, normal prostatic epithelial cells and invasive adenocarcinoma cells obtained from ethanol-fixed, paraffin-embedded tissues. A number of differentially expressed proteins was discovered and identified by mass spectrometry. Immunohistochemical analyses performed on ethanol-fixed tissue sections were in agreement with the proteomic discovery findings. In light of these results, we conclude that ethanol-fixed tissues can be successfully utilized for proteomic analyses.     

Xenopus laevis lectin is localized at several sites in Xenopus oocytes, eggs, and embryos

The endogenous lectin of Xenopus laevis oocytes, unfertilized eggs, and blastula-stage embryos was immunohistochemically localized using a highly specific antiserum. Each tissue was examined with several techniques, including paraformaldehyde or glutaraldehyde fixation, frozen or plastic sections, and immunofluorescence or immunoperoxidase staining. In oocytes and unfertilized eggs, lectin was detected in association with yolk platelets, cortical granules, and the vitelline envelope. In embryos, cortical granules had disappeared and lectin was found in the cleavage furrows between the embryonic cells. The distribution of the lectin suggests that it plays more than one role in this developing system.     

On the mechanism of side chain oxidation of N-beta-alanyldopamine by cuticular enzymes from Sarcophaga bullata

The metabolism of N-beta-alanyldopamine (NBAD) by Sarcophaga bullata was investigated. Incubation of NBAD with larval cuticular preparations resulted in the covalent bindings of NBAD to the cuticle and generation of N-beta-alanyl-norepinephrine (NBANE) as the soluble product. When the reaction was carried out in presence of a powerful quinone trap viz., N-acetylcysteine, NBANE formation was totally abolished; but a new compound characterized as NBAD-quinone-N-acetylcysteine adduct was generated. These results indicate that NBAD quinone is an obligatory intermediate for the biosynthesis of NBANE in sarcophagid cuticle. Accordingly, phenylthiourea--a well-known phenoloxidase inhibitor--completely inhibited the NBANE production even at 5 microM level. A soluble enzyme isolated from cuticle converted exogenously supplied NBAD quinone to NBANE. Chemical considerations indicated that the enzyme is an isomerase and is converting NBAD quinone to its quinone methide which was rapidly and nonenzymatically hydrated to form NBANE. Consistent with this hypothesis is the finding that NBAD quinone methide can be trapped as beta-methoxy NBAD by performing the enzymatic reaction in 10% methanol. Moreover, when the reaction was carried out in presence of kynurenine, two diastereoisomeric structures of papiliochrome II-(Nar-[alpha-3-aminopropionyl amino methyl-3,4-dihydroxybenzyl]-L-kynurenine) could be isolated as by-products, indicating that the further reactions of NBAD quinone methide with exogenously added nucleophiles are nonenzymatic and nonstereoselective. Based on these results, it is concluded that NBAD is metabolized via NBAD quinone and NBAD quinone methide by the action of phenoloxidase and quinone isomerase respectively. The resultant NBAD quinone methide, being highly reactive, undergoes nonenzymatic and nonstereoselective Michael-1,6-addition reaction with either water (to form NBANE) or other nucleophiles in cuticle to account for the proposed quinone methide sclerotization.     

Glycol Methacrylate in Light Microscopy a Routine Method for Embedding and Sectioning Animal Tissues

Glycol methacrylate (GMA), a water and ethanol miscible plastic, was introduced to histology as an embedding medium for electron microscopy. This medium may be made soft enough for cutting thick sections for routine light microscopy by altering its composition. A procedure for the infiltration, polymerization, and sectioning of animal tissues in GMA for light microscopy is presented which is no more complex than paraffin techniques and which has a number of advantages: (I) The GMA medium is compatible with both aqueous fixatives (formaldehyde, glutaraldehyde, Bouin's, and Zenker's) and non-aqueous fixatixes (Carnoy's, Newcomer's, ethanol, and acetone). (2) Undue solvent extraction of the tissue is avoided because adequate dehydration occurs during infiltration of the embedding medium. Separate dehydration and clearing of the tissue prior to embedding is eliminated. (3) When polymerized, the supporting matrix is firm enough that hard and soft tissues adjacent to one another may be sectioned without distortion. (4) Thermal artifact is reduced to a minimum during polymerization because the temperature of the tissue may be maintained at 0-4 C from fixation through ultraviolet light polymerization of the embedding medium. (5) Shrinkage during polymerization of the embedding medium is minimized by prepolymerization of the medium before use. (6) Sections may be easily cut using conventional steel knives and rotary microtomes at a thickness of 0.5 to 3.0 microns, thus improving resolution compared with routinely thicker paraffin sections. (7) The polymerized GMA medium is porous enough so that staining, auto radiography, and other histological procedure are done without removal of the embedding medium from the sections. A list of these stains and related procedures are included. (8) Enzyme digestion of ultra thin sections of tissue embedded in GMA is common in electron microscopic cyto chemistry. me same digestion techniques appear compatible with the thicker seaions used in light microscopy.