Highly Sensitive and Multiplexed Protein Imaging With Cleavable Fluorescent Tyramide Reveals Human Neuronal Heterogeneity

The ability to comprehensively profile proteins in intact tissues in situ is crucial for our understanding of health and disease. However, the existing methods suffer from low sensitivity and limited sample throughput. To address these issues, here we present a highly sensitive and multiplexed in situ protein analysis approach using cleavable fluorescent tyramide and off-the-shelf antibodies. Compared with the current methods, this approach enhances the detection sensitivity and reduces the imaging time by 1–2 orders of magnitude, and can potentially detect hundreds of proteins in intact tissues at the optical resolution. Applying this approach, we studied protein expression heterogeneity in a population of genetically identical cells, and performed protein expression correlation analysis to identify co-regulated proteins. We also profiled >6,000 neurons in a human formalin-fixed paraffin-embedded (FFPE) hippocampus tissue. By partitioning these neurons into varied cell clusters based on their multiplexed protein expression profiles, we observed different sub-regions of the hippocampus consist of neurons from distinct clusters.

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Highly sensitive in situ proteomics with cleavable fluorescent tyramide reveals human neuronal heterogeneity

10.1101/539106 ◽

2019 ◽

Cited By ~ 1

Author(s):

Renjie Liao ◽

Manas Mondal ◽

Christopher D. Nazaroff ◽

Diego Mastroeni ◽

Paul D. Coleman ◽

...

Keyword(s):

Protein Expression ◽

Expression Profiles ◽

Optical Resolution ◽

Detection Sensitivity ◽

Formalin Fixed Paraffin ◽

Highly Sensitive ◽

Formalin Fixed Paraffin Embedded ◽

Low Sensitivity ◽

Protein Expression Profiles

ABSTRACTThe ability to comprehensively profile proteins in intact tissues in situ is crucial for our understanding of health and disease. However, the existing methods suffer from low sensitivity and limited sample throughput. To address these issues, here we present a highly sensitive and multiplexed in situ protein analysis approach using cleavable fluorescent tyramide and off-the-shelf antibodies. Compared with the current methods, this approach enhances the detection sensitivity and reduces the imaging time by 1-2 orders of magnitude, and can potentially detect hundreds of proteins in intact tissues at the optical resolution. Applying this approach, we studied protein expression heterogeneity in genetically identical cells, and performed expression correlation analysis to identify coregulated proteins. We also profiled >6000 neurons in human formalin-fixed paraffin-embedded (FFPE) hippocampus. By partitioning these neurons into varied cell clusters based on their protein expression profiles, we observed different subregions of the hippocampus consist of neurons from distinct clusters.

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Different HER2 Protein Expression Profiles Aid in the Histologic Differential Diagnosis Between Urothelial Carcinoma In Situ (CIS) and Non-CIS Conditions (Dysplasia and Reactive Atypia) of the Urinary Bladder Mucosa

American Journal of Clinical Pathology ◽

10.1309/ajcpkuz69lxzgfea ◽

2011 ◽

Vol 136 (6) ◽

pp. 881-888 ◽

Cited By ~ 11

Author(s):

Sven Gunia ◽

Stefan Koch ◽

Oliver W. Hakenberg ◽

Matthias May ◽

Christoph Kakies ◽

...

Keyword(s):

Differential Diagnosis ◽

Urinary Bladder ◽

Protein Expression ◽

Urothelial Carcinoma ◽

Carcinoma In Situ ◽

Expression Profiles ◽

Bladder Mucosa ◽

Her2 Protein ◽

Protein Expression Profiles

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Highly Sensitive and Multiplexed In-Situ Protein Profiling with Cleavable Fluorescent Streptavidin

Cells ◽

10.3390/cells9040852 ◽

2020 ◽

Vol 9 (4) ◽

pp. 852 ◽

Cited By ~ 3

Author(s):

Renjie Liao ◽

Thai Pham ◽

Diego Mastroeni ◽

Paul D. Coleman ◽

Joshua Labaer ◽

...

Keyword(s):

Optical Resolution ◽

Protein Detection ◽

Protein Analysis ◽

Protein Profiling ◽

Detection Sensitivity ◽

Layer By Layer ◽

Protein Targets ◽

Highly Sensitive ◽

Wide Range

The 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, we here 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 at least 10-fold, compared with the current in-situ proteomics methods. After imaging, the fluorophore and the biotin 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 profiled in individual cells at the optical resolution. Applying this approach, we have demonstrated that multiple proteins are unambiguously detected in the same set of cells, regardless of the protein analysis order. We have also shown that this method can be successfully applied to quantify proteins in formalin-fixed paraffin-embedded (FFPE) tissues.

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IHC versus FISH for the detection of ERG rearrangements in prostate cancer.

Journal of Clinical Oncology ◽

10.1200/jco.2015.33.7_suppl.102 ◽

2015 ◽

Vol 33 (7_suppl) ◽

pp. 102-102

Author(s):

Ekaterina Pestova ◽

Ying Zhang ◽

William Barbanera ◽

Kelly A. Kaiser-Walters ◽

Jing Du ◽

...

Keyword(s):

Protein Expression ◽

Prognostic Significance ◽

Fish Analysis ◽

Formalin Fixed Paraffin ◽

Prognostic Utility ◽

Formalin Fixed Paraffin Embedded ◽

Prostate Epithelium ◽

Benign Prostate ◽

Erg Rearrangement

102 Background: ERG rearrangements are present in about 50% of all prostatic adenocarcinomas (CaP). Using ERG break-apart fluorescence in situ hybridization (FISH) probes, several classes of ERG rearrangement patterns have been described: Edel, Esplit, and 2+Edel. Class ERG 2+Edel was reported to be associated with lethal disease and significantly worse clinical outcome in CaP, while prognostic significance of other classes of ERG rearrangements and of ERG protein expression remains controversial. In this study, we found that IHC cannot discriminate between various forms of ERG rearrangements including 2+Edel, while FISH can. Methods: Formalin fixed paraffin embedded (FFPE) histological specimens from 52 Radical Prostatectomy (RP) cases were used. FISH analysis was performed using Abbott Molecular dual-color break-apart probes for the 5' and 3' regions of the ERG gene. Percent of overall ERG abnormalities and percent of Class 2+Edel per specimen were calculated. ERG expression was evaluated using DAKO FLEX Monoclonal Rabbit Anti-Human ERG Clone EP111. For each slide, H scores (H score =intensity X percentage of positive cells) from both tumor areas and benign prostate epithelium areas were assigned. The IHC score was defined as “H score from tumor minus H score from benign prostate epithelium”. IHC score > 0 was considered positive. Statistical analysis was conducted using JMP 10. Results: Of the 52 RP cases, 9 had the ERG 2+Edel rearrangement (cutoff >2%), 25 had Edel or Esplit rearrangements (cutoff >45%), and 27 had no ERG rearrangements by FISH. The overall Percent Agreement (OPA) between the ERG IHC and FISH (any class of ERG rearrangements) was 90% with 95% Confidence Interval (CI) (79%, 96%). However, for the ERG 2+Edel group, the OPA between the ERG IHC and FISH 2+Edel was 71%, and 62% of IHC positives were false positive. Thus IHC does not discriminate between the 2+Edel, Esplit or Edel forms of ERG while FISH identifies each class of rearrangement. Conclusions: IHC assay allows the detection of overall ERG abnormalities, however it does not discriminate ERG Class 2+Edel, which is consistently reported to be of prognostic significance. Further studies are required to confirm prognostic utility of ERG rearrangements and protein expression in CaP.

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