Modified whole-mount in situ hybridisation and immunohistochemistry protocols without removal of the vitelline membrane in the appendicularian Oikopleura dioica

2017 ◽  
Vol 227 (5) ◽  
pp. 367-374 ◽  
Author(s):  
Takeshi A. Onuma ◽  
Masaki Matsuo ◽  
Hiroki Nishida
Keyword(s):  
In Situ Hybridisation ◽  
Vitelline Membrane ◽  
Oikopleura Dioica ◽  
Whole Mount

218. Expression of Wsb2 in the mouse testis

2005 ◽  
Vol 17 (9) ◽  
pp. 84
Author(s):  
M. Sarraj ◽  
P. J. McClive ◽  
K. L. Loveland ◽  
A. H. Sinclair
Keyword(s):  
Sertoli Cells ◽  
Leydig Cells ◽  
Adult Mouse ◽  
In Situ Hybridisation ◽  
Mouse Testis ◽  
Male Gonad ◽  
Whole Mount ◽  
Mantle Layer ◽  
Pachytene Spermatocytes

We present a detailed study on the expression pattern of Wsb2 in the mouse foetal and adult gonad. Wsb2 expression was analysed during mouse embryogenesis by whole-mount, section in situ hybridisation and immunohistochemistry. Wsb2 was found to be expressed in the developing mouse gonads from 11.5 dpc to 16.5 dpc. Expression is initially equal in both sexes from 10.5 dpc until 12.0 dpc, then it persists in the male gonad. Wsb2 expression was confined to the cords in both Sertoli cell and germ cells. Other sites of Wsb2 embryonic expression were the somites, dorsal root ganglia and the lateral mantle layer of the neural tube. mRNA encoding Wsb2 and Wsb2 protein has been detected in the newborn testis in both gonocytes and Sertoli cells. Wsb2 mRNA in the adult mouse testis was observed in Sertoli cells, spermatogonia, spermatocytes and the corresponding Wsb2 protein expression was in pachytene spermatocytes, round and elongated spermatids, Sertoli cells and Leydig cells. The differential expression of Wsb2 in male versus female embryonic gonads suggests it may play a role in mammalian sex determination during embryonic development and its expression in the first wave of spermatogenesis and in the adult suggests a later role in spermatogenesis.

scholarly journals Medium-Throughput Processing of Whole Mount In Situ Hybridisation Experiments into Gene Expression Domains

PLoS ONE ◽  
2012 ◽  
Vol 7 (9) ◽  
pp. e46658 ◽  
Author(s):  
Anton Crombach ◽  
Damjan Cicin-Sain ◽  
Karl R. Wotton ◽  
Johannes Jaeger
Keyword(s):  
Gene Expression ◽  
In Situ Hybridisation ◽  
Whole Mount

MPP+ IN A ZEBRAFISH MODEL OF GLUCOCEREBROSIDASE 1 DEFICIENCY

2015 ◽  
Vol 86 (11) ◽  
pp. e4.93-e4 ◽  
Author(s):  
Thomas Payne ◽  
Marcus Keatinge ◽  
Marc Da Costa ◽  
Oliver Bandmann
Keyword(s):  
Risk Factors ◽  
Genetic Risk ◽  
In Situ Hybridisation ◽  
Genetic Risk Factors ◽  
Zebrafish Model ◽  
Susceptibility Factor ◽  
Whole Mount ◽  
Glucocerebrosidase Gene ◽  
Proof Of Principle

ObjectiveTo determine if glucocerebrosidase 1 deficiency is a susceptibility factor to 1-methyl-4-phenylpyridinium (MPP+) toxicity using a zebrafish model.Background and hypothesisHeterozygous mutations in the glucocerebrosidase gene (GBA1) are a strong genetic risk factor for Parkinson's disease (PD). Genetic risk factors may increase susceptibility to exogenous neurotoxins. We hypothesized GBA1 haploinsufficiency may increase susceptibility to the classical PD neurotoxin MPTP.MethodsA stable gba1+/− zebrafish line was in-crossed to generate embryos of all genotypes. At 2 days post fertilization (dpf), 100 dechorionated embryos (and 100 controls) were separated into 6 well plates, and exposed to 3 mM MPP+. At 3dpf embryos were fixed in 4% paraformaldehyde. Whole mount in-situ hybridisation was performed using a tyrosine hydroxylase (TH) probe. Embryos were genotyped for gba1 by PCR. TH positive neurons were counted in 10 embryos of each genotype under 20x microscopy.ResultsMPP+ embryos exhibited approximately 25% reduction in TH neurons compared to controls with no differences between genotypes.ConclusionMPP+ exposure results in a loss of TH positive neurons. GBA1 deficiency does not potentiate this. We provide “proof of principle” data for the usefulness of PD mutant zebrafish strains to test possible interactions between PD genetic risk factors and relevant toxins.

RNA and protein localisations of TGF beta 2 in the early mouse embryo suggest an involvement in cardiac development

Development ◽  
1993 ◽  
Vol 117 (2) ◽  
pp. 625-639 ◽  
Author(s):  
M.C. Dickson ◽  
H.G. Slager ◽  
E. Duffie ◽  
C.L. Mummery ◽  
R.J. Akhurst
Keyword(s):  
Cardiac Development ◽  
In Situ Hybridisation ◽  
Outflow Tract ◽  
Tgf Beta ◽  
Atrioventricular Canal ◽  
Whole Mount ◽  
Beta 2 ◽  
Low Levels ◽  
Early Mouse

We have performed a detailed analysis of the localisations of RNAs for TGF beta 2 and beta 3, and of TGF beta 2 protein in mouse embryos from 6.5 to 9.5 days post coitum, using in situ hybridisation and immunohistochemistry on serial sections, and whole-mount in situ hybridisation to complete embryos. TGF beta 3 RNA was not seen in any of the tissue sections, but very low levels of the RNA were seen by whole-mount in situ hybridisation around the outflow tract of the heart at 8.5 days post coitum. TGF beta 2 RNA is expressed at high levels in all cells with the potential to differentiate into cardiomyocytes. Additionally, the foregut endoderm, juxtaposed to the heart, and the neuroepithelium at the rostral extremity of the foregut, express very high levels of TGF beta 2 RNA, between 8.5 and 9.5 days post coitum. As cardiomyogenesis proceeds, TGF beta 2 RNA levels diminishes within the myocytes, with a concomitant increase in staining for TGF beta 2 protein. TGF beta 2 protein staining of cardiomyocytes persists throughout development and in the adult, in the absence of detectable levels of the corresponding RNA. Superimposed upon this myocardial pattern of expression, there is an upregulation of TGF beta 2 RNA in the myocardium of the outflow tract and atrioventricular canal between 8.5 and 9.5 days post coitum, which returns to low levels by 11.5 days post coitum. The results are discussed in terms of a potential role of TGF beta 2 in controlling cardiomyogenesis and in inductive interactions leading to cardiac cushion tissue formation.

Bio-P and non-bio-P bacteria identification by a novel microbial approach

1999 ◽  
Vol 39 (6) ◽  
pp. 13-20 ◽  
Author(s):  
Philip L. Bond ◽  
Jürg Keller ◽  
Linda L. Blackall
Keyword(s):  
In Situ Hybridisation ◽  
Microbial Populations ◽  
Biological Phosphorus Removal ◽  
Gram Positive Bacteria ◽  
P Content ◽  
Identification Of Bacteria ◽  
Widespread Belief ◽  
Biological Phosphorus ◽  
P Removal

Culturing bacteria from activated sludge with enhanced biological phosphorus removal (EBPR) has strongly implicated Acinetobacter with the process. However, using fluorescent in-situ hybridisation (FISH) probing to analyse microbial populations, we have shown evidence opposing this widespread belief. We describe the phosphorus (P) removing performance and microbial population analyses of sludges obtained in a laboratory scale EBPR reactor. Two sludges with extremely high P removing capabilities were examined, the P content of these sludges was 8.6% (P sludge) and 12.3% (S sludge) of the MLSS. Identification of bacteria using FISH probing indicated both sludges were dominated by microbes from the beta proteobacteria and high mol% G+C Gram positive bacteria. Acinetobacter could make up only a small proportion of the cells in these sludges. Sludge with extremely poor P removal (P content of 1.5%, referred to as T sludge) was then generated by reducing the P in the influent. Bacteria resembling the G-bacteria became abundant in this sludge and these were identified using FISH probing. The anaerobic transformations of the T and P sludges correlated well with that of the non-EBPR and EBPR biological models respectively, indicating that bacteria in the T sludge have the potential to inhibit P removal in EBPR systems.

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