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(A) Overview schematic of the PASTA workflow. HRP recruitment including in situ hybridization, unconjugated antibodies, and proximity ligation assays enables tyramine radical formation. These radicals deposit oligos onto nearby proteins in the cells and tissues through covalent binding to tyrosine residues, creating stable protein-oligonucleotide conjugates that resist degradation and enable serial amplification. (B) Concentration-dependent signal amplification of CD3 and Iba1 in <t>FFPE</t> <t>tissues.</t> Images show comparison between unamplified detection (fluorescent oligonucleotide complementary to conjugated oligonucleotide) versus PASTA amplification at increasing tyramine-oligonucleotide concentrations. In the PASTA workflow, antibody-bound oligonucleotides are recognized by complementary oligo-HRP conjugates, which catalyze the deposition of distinct tyramine-oligonucleotide barcodes. Right panels show quantification of log10(fold change) in signal intensity compared to background controls, demonstrating concentration-dependent signal enhancement. Scale bars: 100 µm. (C) Comparison of unamplified versus PASTA-amplified detection for clinically relevant markers (Fas Ligand, CD45RA, PD-L1, CD20, CD11c) on the same tissue section. Violin plots quantify the significant signal enhancement achieved with PASTA amplification for each marker. Scale bars: 100 µm. (D) High-magnification imaging demonstrating spatial resolution and multiplexing capabilities. Left panel: CD11c (yellow), CD3 (magenta), and CD20 (cyan) with mixed amplification (unamplified CD11c, 5 µM PASTA for CD3, 10 µM PASTA for CD20). Right panel: CD45RA (magenta), FasL (yellow), Iba-1 (cyan), and PD-L1 (not colored) with varied PASTA amplification concentrations. Scale bars: 500 µm (main images), 100 µm (insets).
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(A) Overview schematic of the PASTA workflow. HRP recruitment including in situ hybridization, unconjugated antibodies, and proximity ligation assays enables tyramine radical formation. These radicals deposit oligos onto nearby proteins in the cells and tissues through covalent binding to tyrosine residues, creating stable protein-oligonucleotide conjugates that resist degradation and enable serial amplification. (B) Concentration-dependent signal amplification of CD3 and Iba1 in <t>FFPE</t> <t>tissues.</t> Images show comparison between unamplified detection (fluorescent oligonucleotide complementary to conjugated oligonucleotide) versus PASTA amplification at increasing tyramine-oligonucleotide concentrations. In the PASTA workflow, antibody-bound oligonucleotides are recognized by complementary oligo-HRP conjugates, which catalyze the deposition of distinct tyramine-oligonucleotide barcodes. Right panels show quantification of log10(fold change) in signal intensity compared to background controls, demonstrating concentration-dependent signal enhancement. Scale bars: 100 µm. (C) Comparison of unamplified versus PASTA-amplified detection for clinically relevant markers (Fas Ligand, CD45RA, PD-L1, CD20, CD11c) on the same tissue section. Violin plots quantify the significant signal enhancement achieved with PASTA amplification for each marker. Scale bars: 100 µm. (D) High-magnification imaging demonstrating spatial resolution and multiplexing capabilities. Left panel: CD11c (yellow), CD3 (magenta), and CD20 (cyan) with mixed amplification (unamplified CD11c, 5 µM PASTA for CD3, 10 µM PASTA for CD20). Right panel: CD45RA (magenta), FasL (yellow), Iba-1 (cyan), and PD-L1 (not colored) with varied PASTA amplification concentrations. Scale bars: 500 µm (main images), 100 µm (insets).
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(A) Overview schematic of the PASTA workflow. HRP recruitment including in situ hybridization, unconjugated antibodies, and proximity ligation assays enables tyramine radical formation. These radicals deposit oligos onto nearby proteins in the cells and tissues through covalent binding to tyrosine residues, creating stable protein-oligonucleotide conjugates that resist degradation and enable serial amplification. (B) Concentration-dependent signal amplification of CD3 and Iba1 in <t>FFPE</t> <t>tissues.</t> Images show comparison between unamplified detection (fluorescent oligonucleotide complementary to conjugated oligonucleotide) versus PASTA amplification at increasing tyramine-oligonucleotide concentrations. In the PASTA workflow, antibody-bound oligonucleotides are recognized by complementary oligo-HRP conjugates, which catalyze the deposition of distinct tyramine-oligonucleotide barcodes. Right panels show quantification of log10(fold change) in signal intensity compared to background controls, demonstrating concentration-dependent signal enhancement. Scale bars: 100 µm. (C) Comparison of unamplified versus PASTA-amplified detection for clinically relevant markers (Fas Ligand, CD45RA, PD-L1, CD20, CD11c) on the same tissue section. Violin plots quantify the significant signal enhancement achieved with PASTA amplification for each marker. Scale bars: 100 µm. (D) High-magnification imaging demonstrating spatial resolution and multiplexing capabilities. Left panel: CD11c (yellow), CD3 (magenta), and CD20 (cyan) with mixed amplification (unamplified CD11c, 5 µM PASTA for CD3, 10 µM PASTA for CD20). Right panel: CD45RA (magenta), FasL (yellow), Iba-1 (cyan), and PD-L1 (not colored) with varied PASTA amplification concentrations. Scale bars: 500 µm (main images), 100 µm (insets).
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(A) Overview schematic of the PASTA workflow. HRP recruitment including in situ hybridization, unconjugated antibodies, and proximity ligation assays enables tyramine radical formation. These radicals deposit oligos onto nearby proteins in the cells and tissues through covalent binding to tyrosine residues, creating stable protein-oligonucleotide conjugates that resist degradation and enable serial amplification. (B) Concentration-dependent signal amplification of CD3 and Iba1 in <t>FFPE</t> <t>tissues.</t> Images show comparison between unamplified detection (fluorescent oligonucleotide complementary to conjugated oligonucleotide) versus PASTA amplification at increasing tyramine-oligonucleotide concentrations. In the PASTA workflow, antibody-bound oligonucleotides are recognized by complementary oligo-HRP conjugates, which catalyze the deposition of distinct tyramine-oligonucleotide barcodes. Right panels show quantification of log10(fold change) in signal intensity compared to background controls, demonstrating concentration-dependent signal enhancement. Scale bars: 100 µm. (C) Comparison of unamplified versus PASTA-amplified detection for clinically relevant markers (Fas Ligand, CD45RA, PD-L1, CD20, CD11c) on the same tissue section. Violin plots quantify the significant signal enhancement achieved with PASTA amplification for each marker. Scale bars: 100 µm. (D) High-magnification imaging demonstrating spatial resolution and multiplexing capabilities. Left panel: CD11c (yellow), CD3 (magenta), and CD20 (cyan) with mixed amplification (unamplified CD11c, 5 µM PASTA for CD3, 10 µM PASTA for CD20). Right panel: CD45RA (magenta), FasL (yellow), Iba-1 (cyan), and PD-L1 (not colored) with varied PASTA amplification concentrations. Scale bars: 500 µm (main images), 100 µm (insets).
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(A) Overview schematic of the PASTA workflow. HRP recruitment including in situ hybridization, unconjugated antibodies, and proximity ligation assays enables tyramine radical formation. These radicals deposit oligos onto nearby proteins in the cells and tissues through covalent binding to tyrosine residues, creating stable protein-oligonucleotide conjugates that resist degradation and enable serial amplification. (B) Concentration-dependent signal amplification of CD3 and Iba1 in <t>FFPE</t> <t>tissues.</t> Images show comparison between unamplified detection (fluorescent oligonucleotide complementary to conjugated oligonucleotide) versus PASTA amplification at increasing tyramine-oligonucleotide concentrations. In the PASTA workflow, antibody-bound oligonucleotides are recognized by complementary oligo-HRP conjugates, which catalyze the deposition of distinct tyramine-oligonucleotide barcodes. Right panels show quantification of log10(fold change) in signal intensity compared to background controls, demonstrating concentration-dependent signal enhancement. Scale bars: 100 µm. (C) Comparison of unamplified versus PASTA-amplified detection for clinically relevant markers (Fas Ligand, CD45RA, PD-L1, CD20, CD11c) on the same tissue section. Violin plots quantify the significant signal enhancement achieved with PASTA amplification for each marker. Scale bars: 100 µm. (D) High-magnification imaging demonstrating spatial resolution and multiplexing capabilities. Left panel: CD11c (yellow), CD3 (magenta), and CD20 (cyan) with mixed amplification (unamplified CD11c, 5 µM PASTA for CD3, 10 µM PASTA for CD20). Right panel: CD45RA (magenta), FasL (yellow), Iba-1 (cyan), and PD-L1 (not colored) with varied PASTA amplification concentrations. Scale bars: 500 µm (main images), 100 µm (insets).
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Image Search Results


(A) Overview schematic of the PASTA workflow. HRP recruitment including in situ hybridization, unconjugated antibodies, and proximity ligation assays enables tyramine radical formation. These radicals deposit oligos onto nearby proteins in the cells and tissues through covalent binding to tyrosine residues, creating stable protein-oligonucleotide conjugates that resist degradation and enable serial amplification. (B) Concentration-dependent signal amplification of CD3 and Iba1 in FFPE tissues. Images show comparison between unamplified detection (fluorescent oligonucleotide complementary to conjugated oligonucleotide) versus PASTA amplification at increasing tyramine-oligonucleotide concentrations. In the PASTA workflow, antibody-bound oligonucleotides are recognized by complementary oligo-HRP conjugates, which catalyze the deposition of distinct tyramine-oligonucleotide barcodes. Right panels show quantification of log10(fold change) in signal intensity compared to background controls, demonstrating concentration-dependent signal enhancement. Scale bars: 100 µm. (C) Comparison of unamplified versus PASTA-amplified detection for clinically relevant markers (Fas Ligand, CD45RA, PD-L1, CD20, CD11c) on the same tissue section. Violin plots quantify the significant signal enhancement achieved with PASTA amplification for each marker. Scale bars: 100 µm. (D) High-magnification imaging demonstrating spatial resolution and multiplexing capabilities. Left panel: CD11c (yellow), CD3 (magenta), and CD20 (cyan) with mixed amplification (unamplified CD11c, 5 µM PASTA for CD3, 10 µM PASTA for CD20). Right panel: CD45RA (magenta), FasL (yellow), Iba-1 (cyan), and PD-L1 (not colored) with varied PASTA amplification concentrations. Scale bars: 500 µm (main images), 100 µm (insets).

Journal: bioRxiv

Article Title: PASTA: Versatile Tyramine-oligonucleotide Amplification for Multi-modal Spatial Biology

doi: 10.1101/2025.04.30.651463

Figure Lengend Snippet: (A) Overview schematic of the PASTA workflow. HRP recruitment including in situ hybridization, unconjugated antibodies, and proximity ligation assays enables tyramine radical formation. These radicals deposit oligos onto nearby proteins in the cells and tissues through covalent binding to tyrosine residues, creating stable protein-oligonucleotide conjugates that resist degradation and enable serial amplification. (B) Concentration-dependent signal amplification of CD3 and Iba1 in FFPE tissues. Images show comparison between unamplified detection (fluorescent oligonucleotide complementary to conjugated oligonucleotide) versus PASTA amplification at increasing tyramine-oligonucleotide concentrations. In the PASTA workflow, antibody-bound oligonucleotides are recognized by complementary oligo-HRP conjugates, which catalyze the deposition of distinct tyramine-oligonucleotide barcodes. Right panels show quantification of log10(fold change) in signal intensity compared to background controls, demonstrating concentration-dependent signal enhancement. Scale bars: 100 µm. (C) Comparison of unamplified versus PASTA-amplified detection for clinically relevant markers (Fas Ligand, CD45RA, PD-L1, CD20, CD11c) on the same tissue section. Violin plots quantify the significant signal enhancement achieved with PASTA amplification for each marker. Scale bars: 100 µm. (D) High-magnification imaging demonstrating spatial resolution and multiplexing capabilities. Left panel: CD11c (yellow), CD3 (magenta), and CD20 (cyan) with mixed amplification (unamplified CD11c, 5 µM PASTA for CD3, 10 µM PASTA for CD20). Right panel: CD45RA (magenta), FasL (yellow), Iba-1 (cyan), and PD-L1 (not colored) with varied PASTA amplification concentrations. Scale bars: 500 µm (main images), 100 µm (insets).

Article Snippet: The FFPE tissues used in this study were commercially available tonsil tissues (AMSBio, #AMS6022) for and and commercially available HeLa cell pellets (ACDBio, #310045) for .

Techniques: In Situ Hybridization, Ligation, Binding Assay, Amplification, Concentration Assay, Comparison, Marker, Imaging, Multiplexing