Whole-mount Labeling Research Articles

Clearing the path for whole-mount labeling and quantification of neuron- and vessel-density in adipose tissue.

White adipose tissue (WAT) comprises a plethora of cell types beyond adipocytes forming a regulatory network that ensures systemic energy homeostasis. Intertissue communication is facilitated by metabolites and signaling molecules that are spread by vasculature and nerves. Previous works indicated that WAT responds to environmental cues by adapting the abundance of these "communication routes", however, high intra-tissue heterogeneity questions the informative value of bulk or single cell analyses and underscores the necessity of whole-mount imaging. The applicability of whole-mount WAT-imaging is currently limited by two factors: I) Methanol-based tissue clearing protocols restrict the usable antibody portfolio to methanol resistant antibodies and II) The vast amounts of data resulting from 3D imaging of whole-tissue samples require high computational expertise and advanced equipment. Here, we present a protocol for whole-mount WAT clearing, overcoming the constraints of antibody-methanol sensitivity. Additionally, we introduce TiNeQuant (Tissue Network Quantifier) a Fiji tool for automated 3D quantification of neuron- or vascular network density, freely available at https://github.com/SchweigerLab/TiNeQuant. Given TiNeQuants versatility beyond WAT, it simplifies future efforts studying neuronal or vascular alterations in numerous pathologies.

Acetylsalicylic acid interferes with embryonic kidney growth and development by a prostaglandin-independent mechanism.

AIMTo evaluate the effects of the non-selective, non-steroidal anti-inflammatory drug (NSAID) acetylsalicylic acid (ASA), on ex vivo embryonic kidney growth and development.METHODSPairs of fetal mouse kidneys at embryonic day 12.5 were cultured ex vivo in increasing concentrations of ASA (0.04-0.4 mg/mL) for up to 7 d. One organ from each pair was grown in control media and was used as the internal control for the experimental contralateral organ. In some experiments, organs were treated with ASA for 48 h and then transferred either to control media alone or control media containing 10 μmol/L prostaglandin E2 (PGE2) for a further 5 d. Fetal kidneys were additionally obtained from prostaglandin synthase 2 homozygous null or heterozygous (PTGS2-/- and PTGS2-/+) embryos and grown in culture. Kidney cross-sectional area was used to determine treatment effects on kidney growth. Whole-mount labelling to fluorescently detect laminin enabled crude determination of epithelial branching using confocal microscopy.RESULTSIncreasing ASA concentration (0.1, 0.2 and 0.4 mg/mL) significantly inhibited metanephric growth (P < 0.05). After 7 d of culture, exposure to 0.2 mg/mL and 0.4 mg/mL reduced organ size to 53% and 23% of control organ size respectively (P < 0.01). Addition of 10 μmol/L PGE2 to culture media after exposure to 0.2 mg/mL ASA for 48 h resulted in a return of growth area to control levels. Application of control media alone after cessation of ASA exposure showed no benefit on kidney growth. Despite the apparent recovery of growth area with 10 μmol/L PGE2, no obvious renal tubular structures were formed. The number of epithelial tips generated after 48 h exposure to ASA was reduced by 40% (0.2 mg/mL; P < 0.05) and 47% (0.4 mg/mL; P < 0.01). Finally, growth of PTGS2-/- and PTGS2+/- kidneys in organ culture showed no differences, indicating that PTGS2 derived PGE2 may at best have a minor role.CONCLUSIONASA reduces early renal growth and development but the role of prostaglandins in this may be minor.

Onecut transcription factors are required for the second phase of development of the A13 dopaminergic nucleus in the mouse

The A13 dopaminergic nucleus belongs to the incerto-hypothalamic area. It is thought to exert autonomous roles by integrating sensory input to autonomic, neuroendocrine, and motor output. Although its early development has been well characterized, the factors that contribute to later steps of its formation remain unknown. Transcription factors of the Onecut family have been detected in the A13 nucleus, raising the question of possible roles of these factors during A13 development. Using a combination of immunofluorescence analyses on sections and after whole-mount labeling followed by 3D reconstructions, we further characterized the second phase of development of the A13 nucleus in the mouse, described the distribution of the Onecut proteins throughout A13 development, and analyzed the phenotype of this nucleus in single or compound mutant embryos for the Onecut factors. Here we show that A13 development can be divided into two successive phases. First, during radial migration toward the pial surface the A13 cells differentiate into dopaminergic neurons. Second, these cells gather in the vicinity of the third ventricle. Onecut factors are dynamically and differentially expressed in the A13 nucleus during these two phases of development. In Onecut mutant embryos, the A13 neurons differentiate normally but scatter in the diencephalon and fail to properly gather close to the third ventricle. Hence, Onecut factors are markers of the A13 nucleus throughout embryonic development. They are dispensable for the first phase of A13 development but are required for the second phase of development and for maintenance of this nucleus.

Whole-Mount In Situ Hybridization of RNA Probes to Plant Tissues

INTRODUCTIONThe labeling of entire plantlets (whole-mount labeling) is very easy, but it can cause substantial artifacts. Because whole plants are labeled, certain tissues are not readily accessible to the probes, whereas others can often stain nonspecifically. In the hands of the author, for instance, the elongation zone of the root is labeled even in negative controls, whereas mature parts of the roots and hypocotyl tend to be more resistant to probe penetration. Whole-mount labeling should therefore only be used to monitor gene expression in a limited number of organs and tissues--in particular, root meristems, embryos, and very young primordia--and the results should be interpreted with care. A protocol for whole-mount labeling is described here. Positive (e.g., constitutive probe) and negative controls should be included systematically.

Epidermal label-retaining cells: background and recent applications.

Epidermal label-retaining cells (LRC) can be identified by giving neonatal mice repeated injections of 3H-thymidine or 5-bromo-2'-deoxyuridine and then finding the cells that are still labelled in adulthood. Although label retention is simply a marker of the proliferative history of a cell, there is, nevertheless, evidence that it is a characteristic of epidermal stem cells. Here we review the early literature on LRC and then highlight two recent applications. We describe how LRC can be visualized by whole-mount labelling of the epidermis and how purified LRC can be used to screen for markers of the epidermal stem cell compartment.

Manipulation of stem cell proliferation and lineage commitment: visualisation of label-retaining cells in wholemounts of mouse epidermis.

Mammalian epidermis is maintained by stem cells that have the ability to self-renew and generate daughter cells that differentiate along the lineages of the hair follicles, interfollicular epidermis and sebaceous gland. As stem cells divide infrequently in adult mouse epidermis, they can be visualised as DNA label-retaining cells (LRC). With whole-mount labelling, we can examine large areas of interfollicular epidermis and many hair follicles simultaneously, enabling us to evaluate stem cell markers and examine the effects of different stimuli on the LRC population. LRC are not confined to the hair follicle, but also lie in sebaceous glands and interfollicular epidermis. LRC reside throughout the permanent region of the hair follicle, where they express keratin 15 and lie in a region of high alpha6beta4 integrin expression. LRC are not significantly depleted by successive hair growth cycles. They can, nevertheless, be stimulated to divide by treatment with phorbol ester, resulting in near complete loss of LRC within 12 days. Activation of Myc stimulates epidermal proliferation without depleting LRC and induces differentiation of sebocytes within the interfollicular epidermis. Expression of N-terminally truncated Lef1 to block beta-catenin signalling induces transdifferentiation of hair follicles into interfollicular epidermis and sebocytes and causes loss of LRC primarily through proliferation. We conclude that LRC are more sensitive to some proliferative stimuli than others and that changes in lineage can occur with or without recruitment of LRC into cycle.

Study of Cynops pyrrhogaster notochord differentiation using a novel monoclonal antibody.

Two monoclonal antibodies which reacted specifically with the notochord of the early Cynops pyrrhogaster embryo were screened. The antigen molecules were detected within and around the notochord. They were first found mostly between the neural plate and the dorsal part of the notochord in the early neurula (stage 15). They were subsequently detected between the notochord and the somite in the advanced embryo, and they were last detected between the notochord and the underlying endoderm. Whole-mount labeling indicated that the antigen molecules were first detected in the anterior half of the notochord in the early neurula (stage 15). The signals gradually spread along the anterior-posterior axis, especially towards the posterior region. This fact suggests that notochord differentiation progresses from the anterior region which first receives the dorsal mesoderm-inducing signals released horizontally from the lower dorsal marginal zone during early gastrulation. The present study suggested that: (i) notochord differentiation proceeds from the anterior region; and (ii) secretion of the antigen molecules results in the drawing of a boundary between the adjacent tissues.

Whole-mount confocal immunofluorescence of mammalian CNS

Bright-field wholemount labeling techniques applied to the mammalian central nervous system (CNS) offer advantages over conventional methods based on sections since an immediate and three-dimensional view of the stained components is provided. It thereby becomes possible to survey and count large number of cells and fibers in their natural relationships. The ability of confocal laser scanning microscopy to visualize in one focal plane the fluorescence associated with multiple markers could be most valuable by the availability of reliable wholemount fluorescent techniques. Accordingly, based in our previously published bright-field wholemount protocols [Brain Res. Prot. 2 (1998) 165–173], we have devised an effective immmunofluorescence wholemount procedure. We show that reliable wholemount fluorescent staining can be obtained using isolated complete CNS aged up to rat embryonic day 17, with antibodies penetration in the millimeter range. Examples are shown of preparations in which colocalization can be observed in nerve cells of cytoskeletal and calcium-binding proteins.

Mitogen activated protein kinase inhibition by PD98059 blocks nerve growth factor stimulated axonal outgrowth from adult mouse dorsal root ganglia in vitro

Nerve growth factor stimulated axonal outgrowth from explanted mouse dorsal root ganglia is dependent on mitogen activated protein kinase. PD98059 ([2-(2′amino-3′-methoxyphenyl)-oxanaphthalen-4-one]) blocks mitogen activated protein kinase by inhibiting its immediate upstream activator, mitogen activated protein kinase kinase (also known as MEK). Here we used PD98059 to study the role of mitogen activated protein kinase in the axonal outgrowth of adult dorsal root ganglia explants. Whereas PD98059 at 50μM left spontaneous axonal outgrowth unaffected, it markedly inhibited nerve growth factor stimulated axon growth when assessed after two days in culture. A mitogen activated protein kinase assay and immunoblotting using antibodies discriminating between activated and inactivated kinase, both confirmed that PD98059 reduced the amount of activated enzyme in nerve growth factor stimulated preparations, while the total amounts of the kinase remained unchanged. Immunohistochemistry revealed the presence of neuronal mitogen activated protein kinase kinase and mitogen activated protein kinase itself. The latter enzyme was found to be activated in the growing axons, as seen by whole-mount labelling. At the ganglionic level activated mitogen activated protein kinase was preferentially detected in satellite cells.The results show that nerve growth factor stimulated axonal outgrowth in vitro from adult mouse dorsal root ganglia utilizes the mitogen activated protein kinase pathway.

The spatial relationship between stem cells and their progeny in the basal layer of human epidermis: a new view based on whole-mount labelling and lineage analysis.

In order to examine the spatial organisation of stem cells and their progeny in human epidermis, we developed a method for whole-mount epidermal immunofluorescence labelling using high surface beta1 integrin expression as a stem cell marker. We confirmed that there are clusters of high beta1 integrin-expressing cells at the tips of the dermal papillae in epidermis from several body sites, whereas alpha6 integrin expression is more uniform. The majority of actively cycling cells detected by Ki67 or bromodeoxyuridine labelling were found in the beta1 integrin-dull, transit amplifying population and integrin-negative, keratin 10-positive cells left the basal layer exclusively from this compartment. When we examined p53-positive clones in sun-exposed epidermis, we found two types of clone that differed in size and position in a way that was consistent with the founder cell being a stem or transit amplifying cell. The patterning of the basal layer implies that transit amplifying cells migrate over the basement membrane away from the stem cell clusters. In support of this, isolated beta1 integrin-dull keratinocytes were more motile on type IV collagen than beta1 integrin-bright keratinocytes and EGFP-labelled stem cell clones in confluent cultured sheets were compact, whereas transit amplifying clones were dispersed. The combination of whole-mount labelling and lineage marking thus reveals features of epidermal organisation that were previously unrecognised.

Zebrafish immunohistochemistry.

Immunohistochemistry is a powerful technique for determining both the presence of and the subcellular location of proteins within tissues. Zebrafish are particularly amenable to this technique and it is possible to localize proteins both in whole embryos and larvae, as well as sectioned material (Fig. 1). In this chapter, I will describe the basic technique developed for wholemount labeling of zebrafish embryos and variations that are required in specific situations. In addition, for each technique, pitfalls will be highlighted and ways to avoid these suggested.

Procedures for whole-mount immunohistochemistry and in situ hybridization of immature mammalian CNS

Whole-mount labeling techniques for staining in invertebrates or lower vertebrates cannot simply be applied to the mammalian central nervous system (CNS) because of its large size. Such techniques if possible would offer advantages over conventional methods based on sections since an immediate and 3-dimensional view of the stained components in a transparent CNS is provided. It thereby becomes possible to survey and count large number of cells and fibers in their natural relationships. The aim of our experiments is to follow developing and regenerating expression of proteins and mRNAs in the CNS of mouse embryos and newborn opossums ( Monodelphis domestica). Accordingly, we have devised three techniques applicable to whole-mounts: (i) An effective immunohistochemical procedure. This comprises a peroxidase-antiperoxidase method (PAP-WM) based on protocols initially developed for Xenopus embryos and oocytes, including a variation to detect exogenously applied nucleotide analogs such as 5-bromo-2′-deoxyuridine (PAP BrdU-WM). For greater resolution we have introduced a novel gold-silver method (IGSS-WM). (ii) An in situ hybridization procedure (ISH PAP-WM) which combines PAP-WM with protocols described for Xenopus. (iii) A deconvolution (optical sectioning) procedure which improves resolution for bright-field microscopy. We show that reliable whole-mount staining can be obtained using isolated CNS aged up to mouse embryonic day 17 and newborn opossum up to 15 days. Examples are shown of preparations in which one can directly localize nerve cells containing neurotransmitters, cytoskeletal proteins, nucleotide analogs and growth factor messages.