Fluorescence detection of protein clusters in individual cells and tissue sections by using toponome imaging system: sample preparation and measuring procedures

Friedenberger, Manuela; Bode, Marcus; Krusche, A.; Schubert, Walter

doi:10.1038/nprot.2007.320

Cited by 81 publications

(132 citation statements)

References 14 publications

Supporting

Mentioning

131

Contrasting

Order By: Relevance

“…MELC-MELC robot technology involved validated distinct hardware and software components, as described earlier (23,24). After FRET imaging, cells of interest were localized using the square identification number of the coverslips, the YFP fluorescence and the phase contrast images.…”

Section: Methodsmentioning

confidence: 99%

“…A postbleaching image was taken and subtracted from the image taken from the following epitope. Antibody labeling with FITC was performed as described previously (23,24) and signals were validated using conventional immunohistochemistry.…”

Section: Methodsmentioning

confidence: 99%

“…MELC is an automated validated imaging technology that allows the consecutive detection of a large number of fluorophore-labeled primary antibodies on the same sample (23,24,34). Briefly, in this method the application of each antibody is followed by a wash and an imaging step.…”

Section: S1p Decreases Camp Synthesis In Excitatory Spinal Cord Neurons-mentioning

confidence: 99%

See 2 more Smart Citations

Sphingosine 1-Phosphate Modulates Spinal Nociceptive Processing

Coste

Brenneis

Linke

et al. 2008

Journal of Biological Chemistry

View full text Add to dashboard Cite

Sphingosine 1-Phosphate (S1P) modulates various cellular functions such as apoptosis, cell differentiation, and migration. Although S1P is an abundant signaling molecule in the central nervous system, very little is known about its influence on neuronal functions. We found that S1P concentrations were selectively decreased in the cerebrospinal fluid of adult rats in an acute and an inflammatory pain model. Pharmacological inhibition of sphingosine kinases (SPHK) decreased basal pain thresholds and SphK2 knock-out mice, but not SphK1 knock-out mice, had a significant decrease in withdrawal latency. Intrathecal application of S1P or sphinganine 1-phosphate (dihydro-S1P) reduced the pain-related (nociceptive) behavior in the formalin assay. S1P and dihydro-S1P inhibited cyclic AMP (cAMP) synthesis, a key second messenger of spinal nociceptive processing, in spinal cord neurons. By combining fluorescence resonance energy transfer (FRET)-based cAMP measurements with Multi Epitope Ligand Cartography (MELC), we showed that S1P decreased cAMP synthesis in excitatory dorsal horn neurons. Accordingly, intrathecal application of dihydro-S1P abolished the cAMP-dependent phosphorylation of NMDA receptors in the outer laminae of the spinal cord. Taken together, the data show that S1P modulates spinal nociceptive processing through inhibition of neuronal cAMP synthesis.The bioactive sphingolipid metabolite sphingosine 1-phosphate (S1P) 2 is synthesized by phosphorylation of sphingosine by sphingosine kinases (SPHK) in a wide variety of cell types in response to extracellular stimuli such as nerve growth factor or vascular endothelial growth factor. S1P modulates diverse cellular functions such as apoptosis, cell differentiation, and migration either through the activation of a family of five G-protein-coupled receptors (S1P 1-5 ) or by acting as an intracellular second messenger (1-3). In the central nervous system S1P has been shown to be released by cerebellar granule cells and astrocytes (4 -6). Regarding its function in the central nervous system it is well known that S1P promotes survival of neurons and astrocytes, induces proliferation of neural progenitor cells and astrocytes, and mediates nerve growth factor (NGF)-stimulated neurite outgrowth (2, 3, 7). However, very little is known about the role of S1P in the regulation of neuronal excitability and the mechanisms that mediate these S1P functions. Recently whole cell patch clamp recordings revealed that loss of S1P 2 leads to a large increase in the excitability of neocortical pyramidal neurons (8), suggesting an inhibitory effect of S1P 2 on neuronal excitability. On the other hand, several in vitro studies show that S1P can also increase neuronal functions. For example, S1P receptor activation augments glutamate secretion in hippocampal neurons (9) and elevated intracellular S1P concentrations enhance the spontaneous neurotransmitter release at neuromuscular junctions (10). Furthermore, intra-as well as extracellularly applied S1P facilitated the NGF-induced increas...

show abstract

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Sphingosine 1-Phosphate Modulates Spinal Nociceptive Processing

Coste

Brenneis

Linke

et al. 2008

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…Described previously, the multi-epitope-ligand cartography (MELC) 8,9 , is one of these bleaching techniques, that is capable to map the location of different proteins in one sample of cells or tissues using sequential rounds of fluorescent detection 10 . In each cycle, a couple of antibodies are added; phase contrast and fluorescence images then are acquired by a high-sensitivity "charged Coupled Device", a sensor used in digital cameras to record images; the sample is washed with phosphate buffer saline and bleaches at the excitation wavelengths; and post bleaching phase contrast and fluorescence images are acquired.…”

Section: Multi-epitope-ligand Cartographymentioning

confidence: 99%

Novel Platforms of Multiplexed Immunofluorescence for Study of Paraffin Tumor Tissues

Parra¹

2017

J Cancer Treat & Diagnosis

View full text Add to dashboard Cite

Multiplexed immunofluorescence (IF) methods to detect simultaneously different molecules are revolutionizing immunohistochemistry (IHC) in the last years. These new technologies can be valuable for tumor examination in formalin-fixed paraffin-embedded (FFPE) specimens, and for improved new treatment discoveries and translational cancer studies. The aim of this minireview is to highlight the recent methodologies that using multiplexed IF to study simultaneous proteins identification in FFPE tumor tissues to clinical research and potential translational analysis. Multiplexed IF methods, which permit the identification of up to 4 proteins at the same time, have been increased in the last years the abilities of study cells by cells and their spatial distribution in several tumor tissues. Although, most of the old platforms are not more used after the powerful multiplex IHC methods are continue growing, the basis of these old methodologies have helped to improve the new technologies. Associated with image analysis software's these technologies can be improved to performance high throughput assay to study these specimens. Each multiplexed IF technique, detailed herein, is associated with important advantages in cancer study as well as translational research studies.

show abstract

“…Methods for erasing the signals have included low-pH antibody elutions, high-temperature fluorophore denaturation, antibody stripping, and photobleaching. [9][10][11][12][13][14][15][16] Recently, a system ('MultiOmyxt') using dyecycling has been commercialized by GE Healthcare and deployed for a few clinical indications. 17 Because of the serial steps that have to be performed, the throughput is relatively low, and as with all complicated procedures, quality assurance or validation remains a challenge.…”

mentioning

confidence: 99%

Immunohistochemistry and mass spectrometry for highly multiplexed cellular molecular imaging

Levenson

Borowsky

Angelo

2015

Laboratory Investigation

View full text Add to dashboard Cite

The role of immunohistochemistry (IHC) in the management of cancer has expanded to provide improved diagnostic classification, as well as guidance on disease prognosis, therapy, and relapse. These new tasks require evaluation of an increasing number of protein targets; however, conventional multiplexing, usually achieved using serial tissue sections stained for a single analyte per slide, can exhaust small biopsy specimens, complicate slide-to-slide protein expression correlation, and leave insufficient material for additional molecular assays. A new approach, mass spectrometry immunohistochemistry (MSIHC), compatible with high levels of target multiplexing and suitable for use on formalin-fixed, paraffin-embedded samples can circumvent many of these issues. The strategy employs antibodies that are labeled with elemental mass tags, such as isotopically pure lanthanides not typically found in biological specimens, rather than with typical fluorophores or chromogens. The metal-labeled antibodies are then detected in tissue using lasers or ion beams to liberate the tags for subsequent mass spectrometry detection. Within a given multiplexed IHC panel, the metal labels are selected so that their respective masses do not overlap. More than 30 antibodies have been imaged simultaneously, and up to 100 antibodies could potentially be detected at once if the full available mass spectrum is deployed. MSIHC has a number of advantages over conventional IHC techniques. Background due to autofluorescence is absent and the dynamic range is 10 5 , exceeding immunofluorescence and chromogenic IHC by 100-fold and 1000-fold, respectively. Detection of labeled primary antibodies improves assay linearity over both chromogenic and fluorescent IHC. Multiplexed mass-tagged antibodies incubated simultaneously with tissue do not appear to cross-interfere, and because the mass tags do not degrade, samples are stable indefinitely. The imaging resolution of multiplexed ion-beam imaging can be better than light microscopy. With appropriate instrumentation, MSIHC has the potential to transform research and clinical pathology practice.

show abstract

Fluorescence detection of protein clusters in individual cells and tissue sections by using toponome imaging system: sample preparation and measuring procedures

Cited by 81 publications

References 14 publications

Sphingosine 1-Phosphate Modulates Spinal Nociceptive Processing

Sphingosine 1-Phosphate Modulates Spinal Nociceptive Processing

Novel Platforms of Multiplexed Immunofluorescence for Study of Paraffin Tumor Tissues

Immunohistochemistry and mass spectrometry for highly multiplexed cellular molecular imaging

Contact Info

Product

Resources

About