scholarly journals Highly multiplexedin situprotein imaging with signal amplification by Immuno-SABER

2018 ◽  
Author(s):  
Sinem K. Saka ◽  
Yu Wang ◽  
Jocelyn Y. Kishi ◽  
Allen Zhu ◽  
Yitian Zeng ◽  
...  
Keyword(s):  
Exchange Reaction ◽  
Signal Amplification ◽  
Cultured Cells ◽  
Molecular Organization ◽  
Tissue Imaging ◽  
Enzymatic Reactions ◽  
Multiple Targets ◽  
Standard Equipment ◽  
Major Barrier

AbstractProbing the molecular organization of tissues requiresin situanalysis by microscopy. However current limitations in multiplexing, sensitivity, and throughput collectively constitute a major barrier for comprehensive single-cell profiling of proteins. Here, we report Immunostaining with Signal Amplification By Exchange Reaction (Immuno-SABER), a rapid, highly multiplexed signal amplification method that simultaneously tackles these key challenges. Immuno-SABER utilizes DNA-barcoded antibodies and provides a method for highly multiplexed signal amplification via modular orthogonal DNA concatemers generated by Primer Exchange Reaction. This approach offers the capability to preprogram and control the amplification level independently for multiple targets withoutin situenzymatic reactions, and the intrinsic scalability to rapidly amplify and image a large number of protein targets. We validated our approach in diverse sample types including cultured cells, cryosections, FFPE sections, and whole mount tissues. We demonstrated independently tunable 5-180-fold amplification for multiple targets, covering the full signal range conventionally achieved by secondary antibodies to tyramide signal amplification, as well as simultaneous signal amplification for 10 different proteins using standard equipment and workflow. We further combined Immuno-SABER with Expansion Microscopy to enable rapid and highly multiplexed super-resolution tissue imaging. Overall, Immuno-SABER presents an effective and accessible platform for rapid, multiplexed imaging of proteins across scales with high sensitivity.

Modular Simulation to Determine the Optimal Operating Policy of a Batch Reactor for the Enzymatic Fructose Reduction to Mannitol with the in situ Continuous Enzymatic Regeneration of the NAD Cofactor

2017 ◽  
Vol 68 (9) ◽  
pp. 2196-2203 ◽  
Author(s):  
Mara Crisan ◽  
Gheorghe Maria
Keyword(s):  
Batch Reactor ◽  
Production Capacity ◽  
Optimization Procedure ◽  
Enzymatic Reactions ◽  
Mannitol Dehydrogenase ◽  
Optimal Operating ◽  
Operating Policy ◽  
Enzymatic Reduction ◽  
Optimal Operating Policy

Novel coupled enzymatic systems reported important applications in the industrial bio-catalysis. Multi-enzymatic reactions can successfully replace complex chemical syntheses, using milder reaction conditions, and generating less waste. For such systems acting simultaneously, the model-based engineering calculations (design, reactor operation optimization) are difficult tasks, because they must account for interacting reactions, differences in enzymes optimal activity domains and deactivation kinetics. The determination of the optimal operating mode (enzyme ratios, enzyme feeding policy, temperature, pH) often turns into a difficult multi-objective optimization problem with multiple constraints to be solved for every particular system. The paper focuses on applying a modular screening procedure that can identify the optimal operating policy of an enzymatic reactor, which minimizes the enzyme consumption, given the process kinetic model, and an imposed production capacity. Following an optimization procedure, the process effectiveness is evaluated in a systematic approach, by including simple batch reactor (BR), batch with intermittent addition of the key-enzyme following certain optimal policies (BRP). Exemplification is made for the case of the enzymatic reduction of D-fructose to mannitol by using suspended MDH (mannitol dehydrogenase) and NADH (Nicotinamide adenine dinucleotide) cofactor, with the in-situ continuous regeneration of the cofactor by the expense of formate degradation in the presence of suspended FDH (Formate dehydrogenase).

Insight into the enhanced photocatalytic properties of AgBr/Ag4P2O7 composites synthesized via in situ ion exchange reaction

2021 ◽  
Vol 9 (1) ◽  
pp. 104889
Author(s):  
Wyllamanney da S. Pereira ◽  
Fabrício B. Destro ◽  
Cipriano B. Gozzo ◽  
Edson R. Leite ◽  
Júlio C. Sczancoski
Keyword(s):  
Ion Exchange ◽  
Exchange Reaction ◽  
Photocatalytic Properties ◽  
Ion Exchange Reaction ◽  
Insight Into

scholarly journals Detection of v- and c-erbB oncogene mRNA in cultured cells by in situ hybridization.

1990 ◽  
Vol 52 (1) ◽  
pp. 175-178
Author(s):  
Toshio IKEDA ◽  
Yasuhiro YOSHIKAWA ◽  
Kazuya YAMANOUCHI
Keyword(s):  
In Situ Hybridization ◽  
Cultured Cells

Methodologies for specific intron and exon RNA localization in cultured cells by haptenized and fluorochromized probes

1993 ◽  
Vol 104 (4) ◽  
pp. 1187-1197 ◽  
Author(s):  
R.W. Dirks ◽  
F.M. van de Rijke ◽  
S. Fujishita ◽  
M. van der Ploeg ◽  
A.K. Raap
Keyword(s):  
In Situ Hybridization ◽  
Cell Lines ◽  
Cultured Cells ◽  
Direct Detection ◽  
Housekeeping Genes ◽  
Housekeeping Gene ◽  
Immediate Early ◽  
High Signal ◽  
Pretreatment Conditions

We have determined optimal conditions for the detection of mRNA sequences in cultured cells by nonradioactive in situ hybridization. For this purpose a number of different cell lines have been used: rat 9G cells for the detection of human cytomegalovirus immediate early mRNA, and HeLa as well as 5637 carcinoma cells for the detection of housekeeping gene mRNAs. Extensive optimization of fixation and pretreatment conditions revealed that most intense hybridization signals are obtained when cells are grown on glass microscope slides, fixed with a mixture of formaldehyde and acetic acid, pretreated with pepsin and denatured prior to hybridization. In addition, we also studied the potential of fluorochromized probes for the direct detection of multiple RNA sequences. The optimized in situ hybridization procedure revealed that immediate early mRNA transcripts are, in addition to a cytoplasmic localization, localized within nuclei of rat 9G cells. Double hybridization experiments showed that intron and exon sequences colocalize within the main nuclear signal. In addition, the presence of small, intron-specific, fluorescent spots scattered around the main nuclear signals indicates that intron sequences which are spliced out can be visualized. Additional information about the functioning of cells could be obtained by the detection of mRNA simultaneously with bromodeoxyuridine, incorporated during S-phase, or its cognate protein. The sensitivity of these methods is such that mRNAs of abundantly expressed housekeeping genes can be detected in a variety of cell lines with high signal to noise ratios.

scholarly journals Scrapie prion proteins accumulate in the cytoplasm of persistently infected cultured cells.

1990 ◽  
Vol 110 (6) ◽  
pp. 2117-2132 ◽  
Author(s):  
A Taraboulos ◽  
D Serban ◽  
S B Prusiner
Keyword(s):  
Cultured Cells ◽  
Prion Diseases ◽  
Neuroblastoma Cells ◽  
Brain Cell ◽  
Cellular Prion Protein ◽  
Intracellular Accumulation ◽  
Golgi Stack ◽  
Glycosyl Phosphatidylinositol ◽  
The Central Nervous System

The cellular prion protein (PrPC) is a sialoglycoprotein anchored to the external surface of cells by a glycosyl phosphatidylinositol moiety. During scrapie, an abnormal PrP isoform designated PrPSc accumulates, and much evidence argues that it is a major and necessary component of the infectious prion. Based on the resistance of native PrPSc to proteolysis and to digestion with phosphatidylinositol-specific phospholipase C as well as the enhancement of PrPSc immunoreactivity after denaturation, we devised in situ immunoassays for the detection of PrPSc in cultured cells. Using these immunoassays, we identified the sites of PrPSc accumulation in scrapie-infected cultured cells. We also used these immunoassays to isolate PrPSc-producing clones from a new hamster brain cell line (HaB) and found an excellent correlation between their PrPSc content and prion infectivity titers. In scrapie-infected HaB cells as well as in scrapie-infected mouse neuroblastoma cells, most PrPSc was found to be intracellular and most localized with ligands of the Golgi marker wheat germ agglutinin. In one scrapie-infected HaB clone, PrPSc also localized extensively with MG-160, a protein resident of the medial-Golgi stack whereas this colocalization was not observed in another subclone of these cells. Whether the sites of intracellular accumulation of PrPSc are limited to a few subcellular organelles or they are highly variable remains to be determined. If the intracellular accumulation of PrPSc is found in the cells of the central nervous system, then it might be responsible for the neuronal dysfunction and degeneration which are cardinal features of prion diseases.

scholarly journals Highly sensitive and multiplexed in situ protein profiling with cleavable fluorescent streptavidin

2019 ◽  
Author(s):  
Renjie Liao ◽  
Diego Mastroeni ◽  
Paul D. Coleman ◽  
Jia Guo
Keyword(s):  
Signal Amplification ◽  
Individual Cell ◽  
Protein Detection ◽  
Protein Profiling ◽  
Detection Sensitivity ◽  
Layer By Layer ◽  
Protein Targets ◽  
Highly Sensitive ◽  
Wide Range

AbstractThe ability to perform highly sensitive and multiplexed in situ protein analysis is crucial to advance our understanding of normal physiology and disease pathogenesis. To achieve this goal, here we develop an approach using cleavable biotin conjugated antibodies and cleavable fluorescent streptavidin (CFS). In this approach, protein targets are first recognized by the cleavable biotin labeled antibodies. Subsequently, CFS is applied to stain the protein targets. Though layer-by-layer signal amplification using cleavable biotin conjugated orthogonal antibodies and CSF, the protein detection sensitivity can be enhanced by at least 10 fold, compared with the existing methods. After imaging, the fluorophores and the biotins unbound to streptavidin are removed by chemical cleavage. The leftover streptavidin is blocked by biotin. Upon reiterative analysis cycles, a large number of different proteins with a wide range of expression levels can be unambiguously detected in individual cell in situ.

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