Multiplexed In Situ Protein Profiling with High-Performance Cleavable Fluorescent Tyramide

Pham, Thai H.; Liao, Renjie; LaBaer, Joshua; Guo, Jia

doi:10.3390/molecules26082206

Cited by 7 publications

(13 citation statements)

References 56 publications

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“…Subsequently, the fluorophores were cleaved using a mild chemical reaction with 1,3,5-triaza-7-phosphaadamantane (PTA) and tris(2-carboxyethyl)phosphine (TCEP). Following signal removal, almost all the staining signals were erased, confirming the high cleavage efficiency of the CFT as we reported before [22]. We also documented that this chemical reaction does not damage the epitope integrity, which allows other proteins to be accurately profiled in later cycles.…”

Section: Efficient Antibody Stripping While Preserving Epitope Integritysupporting

confidence: 84%

“…Here we demonstrated that the integrity of the protein epitopes is maintained after at least 20 times of protein stripping. Recently, we also reported that the PTA and TCEP treatment does not damage the epitopes [22]. These results suggest more than 20 analysis cycles can be performed on one tissue sample.…”

Section: Discussionmentioning

confidence: 54%

“…Without signal amplification, the low detection sensitivity of these methods limits their applications to study low-expression proteins or to examine specimens with high autofluorescence, such as formalin-fixed paraffin-embedded (FFPE) tissues [18]. To tackle these issues, several laboratories, including ours, have developed several sensitive and multiplexed protein imaging technologies by signal amplifications with biotin-streptavidin interaction [19], oligonucleotide hybridization [20], and horseradish peroxidase (HRP) [21,22]. However, these methods require a chemical-, oligonucleotide-or HRP-labeled primary antibodies to recognize the protein targets.…”

Section: Introductionmentioning

confidence: 99%

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Ultrasensitive and Multiplexed Protein Imaging with Cleavable Fluorescent Tyramide and Antibody Stripping

Pham

Nazaroff

LaBaer

et al. 2021

IJMS

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Multiplexed single-cell analysis of proteins in their native cellular contexts holds great promise to reveal the composition, interaction and function of the distinct cell types in complex biological systems. However, the existing multiplexed protein imaging technologies are limited by their detection sensitivity or technical demands. To address these issues, here, we develop an ultrasensitive and multiplexed in situ protein profiling approach by reiterative staining with off-the-shelf antibodies and cleavable fluorescent tyramide (CFT). In each cycle of this approach, the protein targets are recognized by antibodies labeled with horseradish peroxidase, which catalyze the covalent deposition of CFT on or close to the protein targets. After imaging, the fluorophores are chemically cleaved, and the antibodies are stripped. Through continuous cycles of staining, imaging, fluorophore cleavage and antibody stripping, a large number of proteins can be quantified in individual cells in situ. Applying this method, we analyzed 20 different proteins in each of ~67,000 cells in a human formalin-fixed paraffin-embedded (FFPE) tonsil tissue. Based on their unique protein expression profiles and microenvironment, these individual cells are partitioned into different cell clusters. We also explored the cell–cell interactions in the tissue by examining which specific cell clusters are selectively associating or avoiding each other.

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Section: Efficient Antibody Stripping While Preserving Epitope Integritysupporting

confidence: 84%

Section: Discussionmentioning

confidence: 54%

Section: Introductionmentioning

confidence: 99%

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Ultrasensitive and Multiplexed Protein Imaging with Cleavable Fluorescent Tyramide and Antibody Stripping

Pham

Nazaroff

LaBaer

et al. 2021

IJMS

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“…Integrated in situ analysis of RNA and proteins in the same specimen is of increasing importance in studies of gene expression regulation [ 29 ] and disease diagnosis [ 30 ]. Our laboratory recently developed cleavable fluorescent probes [ 27 , 31 , 32 , 33 ] for multiplexed protein imaging and demonstrated that these probes enable a large amount of different proteins to be accurately quantified in their native cellular contexts in single cells. To evaluate whether the multiplexed RNA and proteins in situ analysis can be combined in the same tissue using CFT, we stained two proteins and five mRNA sequentially using tyramide-N 3 -Cy5 in a mouse spinal cord tissue ( Figure 7 A).…”

Section: Resultsmentioning

confidence: 99%

Highly Sensitive and Multiplexed In Situ RNA Profiling with Cleavable Fluorescent Tyramide

Lü

LaBaer

Guo

2021

Cells

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Understanding the composition, regulation, and function of complex biological systems requires tools that quantify multiple transcripts at their native cellular locations. However, the current multiplexed RNA imaging technologies are limited by their relatively low sensitivity or specificity, which hinders their applications in studying highly autofluorescent tissues, such as formalin-fixed paraffin-embedded (FFPE) tissues. To address this issue, here we develop a multiplexed in situ RNA profiling approach with a high sensitivity and specificity. In this approach, transcripts are first hybridized by target-specific oligonucleotide probes in pairs. Only when these two independent probes hybridize to the target in tandem will the subsequent signal amplification by oligonucleotide hybridization occur. Afterwards, horseradish peroxidase (HRP) is applied to further amplify the signal and stain the target with cleavable fluorescent tyramide (CFT). After imaging, the fluorophores are chemically cleaved and the hybridized probes are stripped by DNase and formamide. Through cycles of RNA staining, fluorescence imaging, signal cleavage, and probe stripping, many different RNA species can be profiled at the optical resolution. In applying this approach, we demonstrated that multiplexed in situ RNA analysis can be successfully achieved in both fixed, frozen, and FFPE tissues.

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“…TSA and ABC can amplify fluorescence signals more than eighty and sixfold, respectively 9 , 10 . However, these two techniques rely on a single chemistry, and for multiplexed signal amplification, the signal amplification process needs to be repeated multiple times for each signal 7 , 11 . Conversely, oligonucleotide-based signal amplification can simultaneously amplify multiple fluorescence signals with the target proteins labeled with oligonucleotide-labeled antibodies and the hybridization of fluorophore-conjugated complementary oligonucleotides 8 .…”

Section: Introductionmentioning

confidence: 99%

Simultaneous amplification of multiple immunofluorescence signals via cyclic staining of target molecules using mutually cross-adsorbed antibodies

Yeon

Cho

Seo

et al. 2022

Sci Rep

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Amplification of immunofluorescence (IF) signals is becoming increasingly critical in cancer research and neuroscience. Recently, we put forward a new signal amplification technique, which we termed fluorescent signal amplification via cyclic staining of target molecules (FRACTAL). FRACTAL amplifies IF signals by repeatedly labeling target proteins with a pair of secondary antibodies that bind to each other. However, simultaneous amplification of multiple IF signals via FRACTAL has not yet been demonstrated because of cross-reactivity between the secondary antibodies. In this study, we show that mutual cross-adsorption between antibodies can eliminate all forms of cross-reactions between them, enabling simultaneous amplification of multiple IF signals. First, we show that a typical cross-adsorption process—in which an antibody binds to proteins with potential cross-reactivity with the antibody—cannot eliminate cross-reactions between antibodies in FRACTAL. Next, we show that all secondary antibodies used in FRACTAL need to be mutually cross-adsorbed to eliminate all forms of cross-reactivity, and then we demonstrate simultaneous amplification of multiple IF signals using these antibodies. Finally, we show that multiplexed FRACTAL can be applied to expansion microscopy to achieve higher fluorescence intensities after expansion. Multiplexed FRACTAL is a highly versatile tool for standard laboratories, as it amplifies multiple IF signals without the need for custom antibodies.

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Multiplexed In Situ Protein Profiling with High-Performance Cleavable Fluorescent Tyramide

Cited by 7 publications

References 56 publications

Ultrasensitive and Multiplexed Protein Imaging with Cleavable Fluorescent Tyramide and Antibody Stripping

Ultrasensitive and Multiplexed Protein Imaging with Cleavable Fluorescent Tyramide and Antibody Stripping

Highly Sensitive and Multiplexed In Situ RNA Profiling with Cleavable Fluorescent Tyramide

Simultaneous amplification of multiple immunofluorescence signals via cyclic staining of target molecules using mutually cross-adsorbed antibodies

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