Labeling of Ultrathin Resin Sections for Correlative Light and Electron Microscopy

Fabig, Gunar; Kretschmar, Susanne; Weiche, Susanne; Eberle, Dominic; Ader, Marius; Kurth, Thomas

doi:10.1016/b978-0-12-416026-2.00005-4

Cited by 32 publications

(28 citation statements)

References 49 publications

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“…Correlative light and electron microscopy (CLEM) combines fluorescent light microscopic (FLM) labeling with electron microscopy (EM), enabling studies of bulky tissue samples from low to sub-cellular high resolution [17]–[19]. We took advantage of the CLEM flexibility to localize the rare events of PPC integration, and to study the formation of OS at an ultra-structural resolution.…”

Section: Resultsmentioning

confidence: 99%

“…Polymerization occurred via UV-irradiation at −35°C. Ultra-thin sections were used for immunofluorescence and/or immuno-EM [17]–[19], [36]. Sections were blocked with 1% BSA in PBS, incubated with rabbit anti-GFP (TP 401 from Torrey Pines or ab 290 from Abcam) followed by either Alexa488 (or Alexa555) conjugated secondary antibodies or by protein A 10-nm gold, and finally counterstained with DAPI (1 µg/ml).…”

Section: Methodsmentioning

confidence: 99%

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Outer Segment Formation of Transplanted Photoreceptor Precursor Cells

et al. 2012

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Transplantation of photoreceptor precursor cells (PPCs) into the retina represents a promising treatment for cell replacement in blinding diseases characterized by photoreceptor loss. In preclinical studies, we and others demonstrated that grafted PPCs integrate into the host outer nuclear layer (ONL) and develop into mature photoreceptors. However, a key feature of light detecting photoreceptors, the outer segment (OS) with natively aligned disc membrane staples, has not been studied in detail following transplantation. Therefore, we used as donor cells PPCs isolated from neonatal double transgenic reporter mice in which OSs are selectively labeled by green fluorescent protein while cell bodies are highlighted by red fluorescent protein. PPCs were enriched using CD73-based magnetic associated cell sorting and subsequently transplanted into either adult wild-type or a model of autosomal-dominant retinal degeneration mice. Three weeks post-transplantation, donor photoreceptors were identified based on fluorescent-reporter expression and OS formation was monitored at light and electron microscopy levels. Donor cells that properly integrated into the host wild-type retina developed OSs with the formation of a connecting cilium and well-aligned disc membrane staples similar to the surrounding native cells of the host. Surprisingly, the majority of not-integrated PPCs that remained in the sub-retinal space also generated native-like OSs in wild-type mice and those affected by retinal degeneration. Moreover, they showed an improved photoreceptor maturation and OS formation by comparison to donor cells located on the vitreous side suggesting that environmental cues influence the PPC differentiation and maturation. We conclude that transplanted PPCs, whether integrated or not into the host ONL, are able to generate the cellular structure for effective light detection, a phenomenon observed in wild-type as well as in degenerated retinas. Given that patients suffering from retinitis pigmentosa lose almost all photoreceptors, our findings are of utmost importance for the development of cell-based therapies.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Outer Segment Formation of Transplanted Photoreceptor Precursor Cells

et al. 2012

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“…Both methods for on-section immuno-EM-labeling can be correlated with light microscopic stainings, either on (almost) adjacent sections (Fabig et al, 2012;Schwarz & Humbel, 2014) or on the same section mounted on an EM grid (Cortese, Diaspro, & Tacchetti, 2009;Mironov & Beznoussenko, 2012;Sjollema, Schnell, Kuipers, Kalicharan, & Giepmans, 2012;Vicidomini et al, 2008Vicidomini et al, , 2010. The CLEM approach allows the fast identification of structures (e.g., cilia) of interest by fluorescent LM at a superb z-resolution.…”

Section: Visualization Of Neural Progenitor Primary Cilia By Tokuyasumentioning

confidence: 99%

Analysis of primary cilia in the developing mouse brain

Paridaen

Huttner

Wilsch‐Bräuninger

2015

Methods in Cell Biology

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“…Alternatively, the primary anti-GFP antibody was detected with secondary antibodies coupled to ultra small gold particles (Aurion,∼1 nm) followed by silver enhancement using the R-Gent SE/Kit (Aurion) before contrasting. For CLEM, ultrathin cryosections were labelled with chicken anti-GFP antibody (Abcam; ab13970), mouse anti-actin antibody (Cedarlane CLT 9001), rabbit antimouse-IgG antibody, protein A conjugated to 10-nm gold particles, and goat-anti-chicken-IgG conjugated to Alexa Fluor 488 as described previously (Fabig et al, 2012). Sections were analysed on a Philips Morgagni 268 TEM (FEI) at 80 kV.…”

Section: Electron Microscopymentioning

confidence: 99%

Essential role of endocytosis for Interleukin-4 receptor mediated JAK/STAT signalling

Kurgonaite

Gandhi

Kurth

et al. 2015

Journal of Cell Science

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Many important signalling cascades operate through specialized signalling endosomes, but a corresponding mechanism has as yet not been described for hematopoietic cytokine receptors. Based on live-cell affinity measurements, we recently proposed that ligandinduced interleukin-4 receptor (IL-4R) complex formation and thus JAK/STAT pathway activation requires a local subcellular increase in receptor density. Here, we show that this concentration step is provided by the internalization of IL-4R subunits through a constitutive, Rac1-, Pak-and actin-mediated endocytosis route that causes IL-4R subunits to become enriched by about two orders of magnitude within a population of cortical endosomes. Consistently, ligand-induced receptor dimers are preferentially detected within these endosomes. IL-4 signalling can be blocked by pharmacological inhibitors targeting the actin polymerization machinery driving receptor internalization, placing endocytosis unambigously upstream of receptor activation. Taken together, these observations demonstrate a role for endocytosis that is mechanistically distinct from the scaffolding function of signalling endosomes in other pathways.

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Labeling of Ultrathin Resin Sections for Correlative Light and Electron Microscopy

Cited by 32 publications

References 49 publications

Outer Segment Formation of Transplanted Photoreceptor Precursor Cells

Outer Segment Formation of Transplanted Photoreceptor Precursor Cells

Analysis of primary cilia in the developing mouse brain

Essential role of endocytosis for Interleukin-4 receptor mediated JAK/STAT signalling

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