ANTIBODY STAINING AND IN SITU HYBRIDIZATION REVEALS THAT A SUBSET OF NEURONS IN THE RABBIT NEONATAL BRAIN PRODUCE INSULIN

Abstract Background The process of photoreception in most animals depends on the light induced isomerization of the chromophore retinal, bound to rhodopsin. To re-use retinal, the all-trans-retinal form needs to be re-isomerized to 11-cis-retinal, which can be achieved in different ways. In vertebrates, this mostly includes a stepwise enzymatic process called the visual cycle. The best studied re-isomerization system in protostomes is the rhodopsin-retinochrome system of cephalopods, which consists of rhodopsin, the photoisomerase retinochrome and the protein RALBP functioning as shuttle for retinal. In this study we investigate the expression of the rhodopsin-retinochrome system and functional components of the vertebrate visual cycle in a polyplacophoran mollusk, Leptochiton asellus, and examine the phylogenetic distribution of the individual components in other protostome animals. Results Tree-based orthology assignments revealed that orthologs of the cephalopod retinochrome and RALBP are present in mollusks outside of cephalopods. By mining our dataset for vertebrate visual cycle components, we also found orthologs of the retinoid binding protein RLBP1, in polyplacophoran mollusks, cephalopods and a phoronid. In situ hybridization and antibody staining revealed that L. asellus retinochrome is co-expressed in the larval chiton photoreceptor cells (PRCs) with the visual rhodopsin, RALBP and RLBP1. In addition, multiple retinal dehydrogenases are expressed in the PRCs, which might also contribute to the rhodopsin-retinochrome system. Conclusions We conclude that the rhodopsin-retinochrome system is a common feature of mollusk PRCs and predates the origin of cephalopod eyes. Our results show that this system has to be extended by adding further components, which surprisingly, are shared with vertebrates.

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X-gal Staining of Drosophila Embryos Compatible with Antibody Staining or In Situ Hybridization

BioTechniques ◽

10.2144/98246bm03 ◽

1998 ◽

Vol 24 (6) ◽

pp. 918-922 ◽

Cited By ~ 11

Author(s):

Ming-Tsan Su ◽

Krista Golden ◽

Rolf Bodmer

Keyword(s):

In Situ Hybridization ◽

Antibody Staining ◽

Drosophila Embryos

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Extrarenal tissue distribution of CHIP28 water channels by in situ hybridization and antibody staining

AJP Cell Physiology ◽

10.1152/ajpcell.1994.266.4.c893 ◽

1994 ◽

Vol 266 (4) ◽

pp. C893-C903 ◽

Cited By ~ 129

Author(s):

H. Hasegawa ◽

S. C. Lian ◽

W. E. Finkbeiner ◽

A. S. Verkman

Keyword(s):

In Situ Hybridization ◽

Epithelial Cells ◽

Tissue Distribution ◽

Choroid Plexus ◽

Ciliary Body ◽

Fluid Transport ◽

Pancreatic Acini ◽

Antibody Staining ◽

Strong Hybridization

This study is an extension of in situ hybridization experiments showing expression of mRNA encoding CHIP28 in selected epithelial or endothelia in spleen, colon, lung, and eye (H. Hasegawa, R. Zhang, A. Dohrman, and A. S. Verkman. Am. J. Physiol. 264 (Cell Physiol. 33): C237-C245, 1993). Additional tissues from rat were screened by in situ hybridization, and tissues from rat and humans were stained with a polyclonal anti-CHIP28 antibody. Northern blot showed the 2.8-kilobase mRNA encoding CHIP28 in kidney, lung, and heart. In situ hybridization showed strong hybridization in epithelial cells in choroid plexus, iris, ciliary body, and lens and in epithelial and subepithelial layers of trachea. Except for colonic crypts, specific hybridization was not observed in the gastrointestinal tract, liver, thyroid gland, and muscle. Immunoblot of tissues from exsanguinated rats showed immunoreactive CHIP28 protein in kidney, lung, trachea, and heart. In fixed frozen rat and/or human tissues, the anti-CHIP28 antibody stained epithelial cells in kidney proximal tubule and thin limb of Henle, lung alveolus, bronchial mucosa and glands, choroid plexus, ciliary body, iris, lens surface, colonic crypt, sweat gland, pancreatic acini, gallbladder epithelium, and placental syncytial trophoblast cells. Endothelial cells were stained in many tissues. These studies indicate a wide and selective CHIP28 tissue distribution, suggesting an important role for CHIP28 in fluid transport. The absence of CHIP28 in many nonrenal membranes believed to be water permeable suggests the existence of non-CHIP28 water transporters.

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Specific detection of neuronal cell bodies: in situ hybridization with a biotin-labeled neurofilament cDNA probe.

Journal of Histochemistry & Cytochemistry ◽

10.1177/34.7.3458811 ◽

1986 ◽

Vol 34 (7) ◽

pp. 923-926 ◽

Cited By ~ 41

Author(s):

P Liesi ◽

J P Julien ◽

P Vilja ◽

F Grosveld ◽

L Rechardt

Keyword(s):

In Situ Hybridization ◽

Cdna Probe ◽

Neuronal Cell ◽

Dna Probe ◽

Specific Detection ◽

Neurofilament Protein ◽

Antibody Staining ◽

Neuronal Cell Bodies ◽

Specific Mrna

We have used a biotinylated, 300-nucleotide cDNA probe which encodes the 68,000 MW neurofilament protein to detect neurofilament-specific mRNA in situ. The neurofilament message specifically demonstrates the neuronal cell bodies, in contrast to the usual antibody staining which detects their neurites. The hybridization is detected only in neuronal structures. Consequently, detection of the biotinylated neurofilament DNA probe by silver-intensified streptavidin-gold can be specifically used to identify neuronal cell bodies.

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Detection and Differentiation of Chlamydiae by Fluorescence In Situ Hybridization

Applied and Environmental Microbiology ◽

10.1128/aem.68.8.4081-4089.2002 ◽

2002 ◽

Vol 68 (8) ◽

pp. 4081-4089 ◽

Cited By ~ 52

Author(s):

Sven Poppert ◽

Andreas Essig ◽

Reinhard Marre ◽

Michael Wagner ◽

Matthias Horn

Keyword(s):

In Situ Hybridization ◽

Fluorescence In Situ Hybridization ◽

Single Step ◽

Detection Methods ◽

The Novel ◽

Antibody Staining ◽

Uncultured Bacteria ◽

Direct Fluorescence ◽

Probe Set

ABSTRACT Chlamydiae are important pathogens of humans and animals but diagnosis of chlamydial infections is still hampered by inadequate detection methods. Fluorescence in situ hybridization (FISH) using rRNA-targeted oligonucleotide probes is widely used for the investigation of uncultured bacteria in complex microbial communities and has recently also been shown to be a valuable tool for the rapid detection of various bacterial pathogens in clinical specimens. Here we report on the development and evaluation of a hierarchic probe set for the specific detection and differentiation of chlamydiae, particularly C. pneumoniae, C. trachomatis, C. psittaci, and the recently described chlamydia-like bacteria comprising the novel genera Neochlamydia and Parachlamydia. The specificity of the nine newly developed probes was successfully demonstrated by in situ hybridization of experimentally infected amoebae and HeLa 229 cells, including HeLa 229 cells coinfected with C. pneumoniae and C. trachomatis. FISH reliably stained chlamydial inclusions as early as 12 h postinfection. The sensitivity of FISH was further confirmed by combination with direct fluorescence antibody staining. In contrast to previously established detection methods for chlamydiae, FISH was not susceptible to false-positive results and allows the detection of all recognized chlamydiae in one single step.

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