Maintenance of Fluorescence During Paraffin Embedding of Fluorescent Protein-Labeled Specimens

Zhanmu, Ouyang; Zhao, Peilin; Yang, Yang; Yang, Xiaoquan; Gong, Hui; Li, Xiangning

doi:10.3389/fnins.2019.00752

Cited by 17 publications

(17 citation statements)

References 27 publications

(34 reference statements)

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“…A quantitative assessment of immunofluorescence-stained sections was performed for each dilution for each serum pool [ 45 , 46 ]. Ten 40 × fields were randomly photographed under an optical microscope (Leica DM6000B by Leica, Wetzlar, Germany) coupled with a digital camera (Leica DFC450C digital camera by Leica).…”

Section: Methodsmentioning

confidence: 99%

Leishmania spp.-Infected Dogs Have Circulating Anti-Skeletal Muscle Autoantibodies Recognizing SERCA1

et al. 2021

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Leishmania spp. infection is associated with an inflammatory myopathy (IM) in dogs. The pathomechanism underlying this disorder is still elusive, however, the pattern of cellular infiltration and MHC I and II upregulation indicate an immune-mediated myositis. This study aimed to investigate the presence of autoantibodies targeting the skeletal muscle in sera of leishmania-infected dogs and individuate the major autoantigen. We tested sera from 35 leishmania-infected dogs and sera from 10 negative controls for the presence of circulating autoantibodies with indirect immunofluorescence. Immunoblot and mass spectrometry were used to identify the main target autoantigen. Immunocolocalization and immunoblot on immunoprecipitated muscle proteins were performed to confirm the individuated major autoantigen. We identified circulating autoantibodies that recognize skeletal muscle antigen(s) in sera of leishmania-infected dogs. The major antigen was identified as the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 1 (SERCA1). We also found that canine SERCA1 presents several identical traits to the calcium-translocating P-type ATPase of Leishmania infantum. In the present study, we defined circulating anti-SERCA1 autoantibodies as part of the pathogenesis of the leishmania-associated IM in dogs. Based on our data, we hypothesize that antigen mimicry is the mechanism underlying the production of these autoantibodies in leishmania-infected dogs.

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Section: Methodsmentioning

confidence: 99%

Leishmania spp.-Infected Dogs Have Circulating Anti-Skeletal Muscle Autoantibodies Recognizing SERCA1

et al. 2021

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“…It is well suited for immunofluorescence microscopy (Fig. 11B, [Morris, Klanke, Lang, Lim, & Crombleholme, 2010], [Quell et al, 2017]) without extensive optimization (Zhanmu et al, 2019). Furthermore, tdTomato emits more strongly than most other chromophores (Shaner et al, 2004;Shaner et al, 2005) making it a very in-teresting surrogate for cells with minor expression of the target molecule.…”

Section: Strength Of the Reporter Moleculesmentioning

confidence: 99%

Characterization of Anaphylatoxin Receptor Expression and C3a/C5a Functions in Anaphylatoxin Receptor Reporter Mice

et al. 2020

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The anaphylatoxins (AT) C3a and C5a are effector molecules of C3 and C5 exerting multiple biologic functions through binding and activation of their cognate G protein−coupled receptors. C3a interacts with the C3a receptor (C3aR), whereas C5a and its primary degradation product C5a-desArg engage C5aR1 and C5aR2. In the past, analysis of AT expression has been hampered by cross reaction of antibodies designed to recognize the different AT receptors. Furthermore, assessment of effects mediated by cell-specific activation has been difficult. Here, floxed AT receptor reporter mice are described as tools to monitor AT receptor expression in cells and tissues and to study the functions of C3a and C5a by cell-specific deletion of their cognate AT receptors.

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“…Although CX3CR1 +/− mice are often used to represent mice with normal CX3CR1 function, in strict consideration, we chose wild-type mice as the normal control to ensure the complete retention of CX3CR1 function. In order to compensate for the difference of GFP fluorescence between wild-type mice and CX3CR1 −/− mice, we used ethanol dehydration and paraffin embedding to quench GFP fluorescence as previously described [22]. After anesthesia, mice were first transcardially perfused with 20 ml 0.1M pre-cooled phosphate buffered saline (PBS) until colorless liquid drained out of the right auricle.…”

Section: Immunohistochemistrymentioning

confidence: 99%

CX3CR1 modulates cognitive dysfunction induced by sleep deprivation

Xin¹,

Cheng²,

Xie³

et al. 2020

Preprint

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Background Sleep disorder-associated cognitive impairments are often accompanied by impaired synaptic plasticity. In the pathological process of sleep deprivation, microglia, as an important innate immune cell in central nervous system, are involved and mediate the synaptic pruning, in which the CX3C-chemokine receptor 1 receptor (CX3CR1) plays an indispensable role. In this study, the potential role of CX3CR1 in modulating the cognition decline during sleep deprivation was evaluated in terms of microglial neuroinflammation and synaptic pruning. Methods Middle-aged wild type C57BL/6 mice (WT) (13 months) and age-matched CX3CR1 -/- mice were either subjected to sleep deprivation (SD) or allowed normal sleep (S) for 8 h to mimic the pathophysiological changes of middle-aged people after staying up all night in real life. The hippocampus-dependent cognition was assessed by fear conditioning test. Mice brains were extracted for neuroinflammation and synaptic pruning studies. Data were analyzed by Student’s t-test and one-way analysis of variance (one-way ANOVA). Results The CX3CR1 deficiency mice differed from WT mice in many measures. CX3CR1 deficiency prevented SD-induced cognitive impairments, in which the levels of brain-derived neurotrophic factor (BDNF) and phosphorylated cAMP-responsive element binding protein (p-CREB) were markedly increased in the hippocampus. Compared with the CX3CR1 -/- -S group, the CX3CR1 -/- -SD mice reported a markedly decreased microglial density in the dentate gyrus, notably-decreased levels of cellular oncogene fos (c-fos), and pro-inflammatory cytokines and increased levels of anti-inflammatory cytokines in the hippocampus, and a significant increase in the density of spines of the dentate gyrus area. Furthermore, the CX3CR1 -/- -SD group also showed a decreasing expression of microglial phagocytosis-related factors. Conclusion These findings suggest that CX3CR1 deficiency leads to different cerebral behaviors and responses to sleep deprivation. CX3CR1 deletion can alleviate the sleep deprivation-induced cognitive impairment. The inflammation-attenuating activity and the related modification of synaptic pruning are possible mechanism candidates, which indicate CX3CR1 as a candidate therapeutic target for the prevention of the sleep loss-induced cognitive impairments.

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Maintenance of Fluorescence During Paraffin Embedding of Fluorescent Protein-Labeled Specimens

Cited by 17 publications

References 27 publications

Leishmania spp.-Infected Dogs Have Circulating Anti-Skeletal Muscle Autoantibodies Recognizing SERCA1

Leishmania spp.-Infected Dogs Have Circulating Anti-Skeletal Muscle Autoantibodies Recognizing SERCA1

Characterization of Anaphylatoxin Receptor Expression and C3a/C5a Functions in Anaphylatoxin Receptor Reporter Mice

CX3CR1 modulates cognitive dysfunction induced by sleep deprivation

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