scholarly journals High-Resolution Whole-MountIn SituHybridization Using Quantum Dot Nanocrystals

2012 ◽  
Vol 2012 ◽  
pp. 1-9 ◽  
Author(s):  
Andriani Ioannou ◽  
Iro Eleftheriou ◽  
Andrea Lubatti ◽  
Anna Charalambous ◽  
Paris A. Skourides
Keyword(s):  
Expression Patterns ◽  
Intracellular Localization ◽  
Emission Spectra ◽  
Fluorescent Imaging ◽  
Proteinase K ◽  
Fluorescent Detection ◽  
Organic Fluorophores ◽  
Whole Mount ◽  
Increased Sensitivity

The photostability and narrow emission spectra of nanometer-scale semiconductor crystallites (QDs) make them desirable candidates for whole-mount fluorescentin situhybridization to detect mRNA transcripts in morphologically preserved intact embryos. We describe a method for direct QD labeling of modified oligonucleotide probes through streptavidin-biotin and antibody-mediated interactions (anti-FITC and anti-digoxigenin). To overcome permeability issues and allow QD conjugate penetration, embryos were treated with proteinase K. The use of QDs dramatically increased sensitivity of whole-mountin situhybridization (WISH) in comparison with organic fluorophores and enabled fluorescent detection of specific transcripts within cells without the use of enzymatic amplification. Therefore, this method offers significant advantages both in terms of sensitivity, as well as resolution. Specifically, the use of QDs alleviates issues of photostability and limited brightness plaguing organic fluorophores and allows fluorescent imaging of cleared embryos. It also offers new imaging possibilities, including intracellular localization of mRNAs, simultaneous multiple-transcript detection, and visualization of mRNA expression patterns in 3D.

mRNA expression patterns of the IGF system during mouse limb bud development, determined by whole mount in situ hybridization

1998 ◽  
Vol 138 (1-2) ◽  
pp. 151-161 ◽  
Author(s):  
Marjolein van Kleffens ◽  
Cora Groffen ◽  
Roberto R. Rosato ◽  
Stefan M. van den Eijnde ◽  
Johan W. van Neck ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Mrna Expression ◽  
Expression Patterns ◽  
Limb Bud ◽  
Bud Development ◽  
Igf System ◽  
Whole Mount ◽  
Mouse Limb

scholarly journals Cytochrome P450 Expression and Chemical Metabolic Activity before Full Liver Development in Zebrafish

2020 ◽  
Vol 13 (12) ◽  
pp. 456
Author(s):  
Tasuku Nawaji ◽  
Natsumi Yamashita ◽  
Haruka Umeda ◽  
Shuangyi Zhang ◽  
Naohiro Mizoguchi ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
In Situ Hybridization ◽  
Metabolic Activity ◽  
Expression Patterns ◽  
Xenobiotic Metabolism ◽  
Liver Development ◽  
P450 Expression ◽  
Whole Mount ◽  
Metabolic Activities

Zebrafish are used widely in biomedical, toxicological, and developmental research, but information on their xenobiotic metabolism is limited. Here, we characterized the expression of 14 xenobiotic cytochrome P450 (CYP) subtypes in whole embryos and larvae of zebrafish (4 to 144 h post-fertilization (hpf)) and the metabolic activities of several representative human CYP substrates. The 14 CYPs showed various changes in expression patterns during development. Many CYP transcripts abruptly increased at about 96 hpf, when the hepatic outgrowth progresses; however, the expression of some cyp1s (1b1, 1c1, 1c2, 1d1) and cyp2r1 peaked at 48 or 72 hpf, before full liver development. Whole-mount in situ hybridization revealed cyp2y3, 2r1, and 3a65 transcripts in larvae at 55 hpf after exposure to rifampicin, phenobarbital, or 2,3,7,8-tetrachlorodibenzo-p-dioxin from 30 hpf onward. Marked conversions of diclofenac to 4′-hydroxydiclofenac and 5-hydroxydiclofenac, and of caffeine to 1,7-dimethylxanthine, were detected as early as 24 or 50 hpf. The rate of metabolism to 4’-hydroxydiclofenac was more marked at 48 and 72 hpf than at 120 hpf, after the liver had become almost fully developed. These findings reveal the expression of various CYPs involved in chemical metabolism in developing zebrafish, even before full liver development.

scholarly journals Patterns of gene expression in schistosomes: localization by whole mountin situhybridization

Parasitology ◽  
2007 ◽  
Vol 134 (11) ◽  
pp. 1589-1597 ◽  
Author(s):  
G. P. DILLON ◽  
J. C. ILLES ◽  
H. V. ISAACS ◽  
R. A. WILSON
Keyword(s):  
Gene Expression ◽  
Cathepsin L ◽  
Expression Patterns ◽  
Proteinase K ◽  
Fluorescent Substrate ◽  
Rna Probes ◽  
Fast Red ◽  
Proteinase K Treatment

SUMMARYAs a consequence of comprehensive transcriptome analysis followed by sequencing and draft assembly of the genome, the emphasis of schistosome research is shifting from the identification of genes to the characterization of their functions and interactions. Developmental biologists have long used whole mountin situhybridization (WISH) to determine gene expression patterns, as a vital tool for formulating and testing hypotheses about function. This paper describes the application of WISH to the study of gene expression in larval and adult schistosomes. Fixed worms were permeablized by proteinase K treatment for hybridization with digoxygenin-labelled RNA probes, with binding being detected by alkaline phosphatase-coupled anti-digoxygenin antibodies, and BM Purple substrate. Discrete staining patterns for the transcripts of the molecules Sm29, cathepsin L, antigen 10.3 and chorion were observed in the tegument cell bodies, gut epithelium, oesophageal gland and vitelline lobules, respectively, of adult worms. Transcripts of the molecules SGTP4, GP18-22 and cathepsin L were localized to tegument cell bodies and embryonic gut, respectively, of lung schistosomula. We also showed that Fast Red TR fluorescent substrate can refine the pattern of localization permitting use of confocal microscopy. We believe that method of WISH will find broad application, in synergy with other emerging post-genomic techniques, such as RNA interference, to studies focused at increasing our molecular understanding of schistosomes.

Expression Patterns of RNAs for Amelin and Amelogenin in Developing Rat Molars and Incisors

1996 ◽  
Vol 10 (2) ◽  
pp. 195-200 ◽  
Author(s):  
C.D. Fong ◽  
L. Hammarström ◽  
C. Lundmark ◽  
T. Wurtz ◽  
I. Slaby
Keyword(s):  
In Situ Hybridization ◽  
Expression Patterns ◽  
Paraffin Sections ◽  
Hybridization Procedure ◽  
Whole Mount ◽  
First Time ◽  
Developing Rat ◽  
Rat Molars ◽  
Oral Epithelia

We have recently identified a novel RNA sequence in ameloblasts, coding for amelin (Cerny et al., 1996). In the present paper, its expression has been compared with that of amelogenin in developing incisors and molars of rats, by means of in situ hybridization of paraffin sections. The RNAs for both amelin and amelogenin were highly expressed in secretory ameloblasts. The expression of RNA for amelogenin gradually decreased in the post-secretory ameloblasts. In contrast, the RNA expression for amelin remained high in post-secretory ameloblasts up to the stage of fusion between dental and oral epithelia at the time of tooth eruption. We suggest that amelin might be involved in the mineralization of enamel or in the attachment of ameloblasts to the enamel surface. The whole-mount in situ hybridization procedure is described for the first time in dental research. It proved to be a useful method and confirmed the results of the conventional in situ hybridization.

scholarly journals All-age whole mount in situ hybridization to reveal larval and juvenile expression patterns in zebrafish

PLoS ONE ◽  
2020 ◽  
Vol 15 (8) ◽  
pp. e0237167
Author(s):  
Franz Vauti ◽  
Luisa A. Stegemann ◽  
Viktoria Vögele ◽  
Reinhard W. Köster
Keyword(s):  
In Situ Hybridization ◽  
Expression Patterns ◽  
Whole Mount

Heat-Induced Antigen Retrieval Applied in Zebrafish: Whole-Mount In Situ Immunofluorescence Microscopy

2012 ◽  
Vol 18 (3) ◽  
pp. 493-496 ◽  
Author(s):  
Chuang-yu Lin ◽  
Wen-ta Su ◽  
Li-tzu Li
Keyword(s):  
Fluorescent Protein ◽  
Immunofluorescence Microscopy ◽  
Expression Patterns ◽  
Biological Molecules ◽  
Antigen Retrieval ◽  
Zebrafish Embryos ◽  
Fluorescence Dye ◽  
Whole Mount ◽  
Green Fluorescent

AbstractWhole-mount immunofluorescence technique provides a way to reveal integrated expression patterns of biological molecules in individuals. Well-documented morphological preservation ability in biology makes aldehydes the fixative of choice. Cross-linking among biocomponents and aldehydes is the key for maintaining morphology but masks the biological molecules for immunodetection. This study performs an easily accessible method by applying heat-induced retrieval, which can rescue the antigenicity of the proteins and also enhance the labeling sensitivity of the fluorescence dye in overfixed zebrafish embryos. The results show that the immunoreactivities of antibodies to myosin in the muscles, green fluorescent protein in the blood vessels and the nuclei in the cells can be recovered significantly, and the morphology of the zebrafish embryos, even the fragile mutants, is at the same time well maintained. Therefore, we provide a choice for antigen retrieval, which is effective for whole-mount immunofluorescence microscopy.

scholarly journals A powerful and versatile new fixation protocol for immunohistology and in situ hybridization that preserves delicate tissues in planaria

2021 ◽  
Author(s):  
Carlos Guerrero-Hernández ◽  
Viraj Doddihal ◽  
Frederick G. Mann ◽  
Alejandro Sánchez Alvarado
Keyword(s):  
In Situ Hybridization ◽  
Formic Acid ◽  
Fluorescent In Situ Hybridization ◽  
Acid Treatment ◽  
Confocal Imaging ◽  
Proteinase K ◽  
Whole Mount ◽  
Fixation Protocol ◽  
Regeneration Blastema

Whole-mount in situ hybridization (WISH) is a powerful and widely used technique to visualize the expression pattern of genes in different biological systems. Here we describe a new protocol for ISH and immunostaining in the planarian Schmidtea mediterranea. The new Nitric Acid/Formic Acid (NAFA) protocol is compatible with both assays and prevents degradation of the epidermis or blastema. Instead of proteinase K digestion, formic acid treatment is used to permeabilize tissues and preserve antigen epitopes. We show that the NAFA protocol successfully permits development of chromogenic and fluorescent signals in situ, while preserving the anatomy of the animal. Further, the immunostaining of different proteins was compatible with the NAFA protocol following fluorescent in situ hybridization. Finally, we demonstrate with high resolution confocal imaging that the regeneration blastema is preserved when using the new method. This new NAFA protocol will be a valuable technique to study the process of wounding response and regeneration.

scholarly journals Sensitive whole mount in situ localization of small RNAs in plants

2016 ◽  
Author(s):  
Mouli Ghosh Dastidar ◽  
Magdalena Mosiolek ◽  
Michael D Nodine ◽  
Alexis Maizel
Keyword(s):  
Small Rnas ◽  
Direct Detection ◽  
Expression Patterns ◽  
Systematic Investigation ◽  
Indirect Detection ◽  
Whole Mount ◽  
Small Regulatory Rnas ◽  
Regulatory Rnas ◽  
In Situ Detection

Small regulatory RNAs are pivotal regulators of gene expression and play important roles in many plant processes. Although our knowledge of their biogenesis and mode of action has significantly progressed, we comparatively still know little about their biological functions. In particular, knowledge about their spatiotemporal patterns of expression rely on either indirect detection by use of reporter constructs or labor-intensive direct detection by in situ hybridization on sectioned material. None of the current approaches allows for a systematic investigation of small RNAs expression patterns.Here, we present a method for the sensitive in situ detection of micro- and siRNAs in intact plant tissues that utilizes both double-labelled probes and a specific cross linker. We determined the expression patterns of several small RNAs in plant roots and embryos.

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