Journal: bioRxiv

Article Title: Direct measurement of RNA Polymerase II hypertranscription in cancer FFPE samples

doi: 10.1101/2024.02.28.582647

Figure Lengend Snippet: Pathology classification, age and sex were provided by the vendor (BioChain). Each image spans the width of a standard charged microscope slide, where the tissue is visible under the paraffin skin. On-slide RNAPII-Ser5p FFPE-CUTAC was applied to slides in parallel, using a total of four slides each for 100 separate samples in all to produce the data analyzed in this study.

Article Snippet: The following pairs of human tumor and adjacent normal 5 μm tissue sections from single FFPE blocks were purchased from Biochain, Inc: Breast Normal/Tumor cat. no. T8235086PP/PT; Colon Normal/Tumor cat. no. T8235090PP/PT; Kidney Normal/Tumor cat. no. T8235142PP/PT; Liver Normal/Tumor cat. no. T8235149PP/PT; Lung Normal/Tumor cat. no. T8235152PP/PT; Rectum Normal/Tumor cat. no. T8235206PP/PT; Stomach Normal/Tumor cat. no. T8235248PP/PT.

Techniques: Microscopy

Journal: ACS Chemical Biology

Article Title: Multiplex, Quantitative, High-Resolution Imaging of Protein:Protein Complexes via Hybridization Chain Reaction

doi: 10.1021/acschembio.3c00431

Figure Lengend Snippet: Imaging protein:protein complexes in human cells, mouse proT cells, and FFPE human breast tissue sections. (A,B) Imaging β-catenin:E-cadherin target complex in A-431 cells expressing β-catenin and E-cadherin (panel A) or HeLa cells expressing N-cadherin instead of E-cadherin (panel B). (C,D) Imaging RUNX1:PU.1 target complex in Scid.adh.2C2 mouse proT cells retrovirally transduced with a PU.1-expressing vector (panel C) or an empty vector (panel D). (E,F) Imaging β-catenin:E-cadherin target complex in 5 μm FFPE human breast tissue sections from the same patient: normal (panel E) or invasive lobular carcinoma (panel F). All panels: confocal image; single optical section; 0.18 × 0.18 × 0.8 μm pixels (panels A–D) or 0.57 × 0.57 × 3.3 μm pixels (panels E,F). Signal-to-backround ratio for each row (mean ± SEM for representative regions of N = 3 replicate samples). See sections S2.2–S2.4 for additional data.

Article Snippet: HCR imaging of protein:protein complexes was performed in 5 μm FFPE normal human breast tissue sections (Acepix Biosciences, HuN-06-0027) and 5 μm FFPE invasive lobular carcinoma human breast tissue sections (Acepix Biosciences, HuC-06-0101) from the same patient using the protocol detailed in section S1.11 .

Techniques: Imaging, Expressing, Transduction, Plasmid Preparation

Journal: ACS Chemical Biology

Article Title: Multiplex, Quantitative, High-Resolution Imaging of Protein:Protein Complexes via Hybridization Chain Reaction

doi: 10.1021/acschembio.3c00431

Figure Lengend Snippet: qHCR imaging: relative quantitation of protein:protein complexes with subcellular resolution in an anatomical context. (A) Two-channel redundant detection of a protein:protein complex: each target protein is detected by an unlabeled primary antibody probe and two batches of secondary antibody probes that interact with orthogonal proximity probes to colocalize full HCR initiators that trigger orthogonal spectrally distinct HCR amplifiers (Ch1, Alexa546; Ch2, Alexa647). (B) Two-channel confocal images; single optical sections. Top: β-catenin:E-cadherin complex in A-431 cells (0.18 × 0.18 × 0.8 μm pixels). Bottom: β-catenin:E-cadherin complex in a 5 μm FFPE normal human breast tissue section (0.57 × 0.57 × 3.3 μm pixels). (C) High accuracy and precision for protein:protein relative quantitation in an anatomical context. Highly correlated normalized signal (Pearson correlation coefficient, r ) for subcellular voxels in the indicated regions in panel B. Top: 2.0 × 2.0 × 0.8 μm voxels. Bottom: 2.0 × 2.0 × 3.3 μm voxels. Accuracy: linearity with zero intercept. Precision: scatter around the line. See section S2.6 for additional data.

Article Snippet: HCR imaging of protein:protein complexes was performed in 5 μm FFPE normal human breast tissue sections (Acepix Biosciences, HuN-06-0027) and 5 μm FFPE invasive lobular carcinoma human breast tissue sections (Acepix Biosciences, HuC-06-0101) from the same patient using the protocol detailed in section S1.11 .

Techniques: Imaging, Quantitation Assay

Journal: Development (Cambridge, England)

Article Title: Hybridization chain reaction enables a unified approach to multiplexed, quantitative, high-resolution immunohistochemistry and in situ hybridization

doi: 10.1242/dev.199847

Figure Lengend Snippet: Multiplexed protein imaging via HCR 1°IHC using initiator-labeled primary antibody probes and simultaneous HCR signal amplification for all targets. (A) Two-stage HCR 1°IHC protocol. Detection stage: initiator-labeled primary antibody probes bind to protein targets; wash. Amplification stage: initiators trigger self-assembly of fluorophore-labeled HCR hairpins into tethered fluorescent amplification polymers; wash. (B) Multiplexing timeline. The same two-stage protocol is used independent of the number of target proteins. (C) Confocal image of 3-plex protein imaging in mammalian cells on a slide; 0.2×0.2 µm pixels; maximum intensity z -projection. Target proteins: HSP60 (Alexa488), GM130 (Alexa647) and SC35 (Alexa546). Sample: HeLa cells. (D) Epifluorescence image of 4-plex protein imaging in FFPE mouse brain sections; 0.3×0.3 µm pixels. Target proteins: TH (Alexa488), GFAP (Alexa546), MBP (Alexa647) and MAP2 (Alexa750). (E) Zoom of indicated region in D. Sample: FFPE C57BL/6 mouse brain section (coronal); 5 µm thickness. See section S5.2 of the supplementary information for additional data.

Article Snippet: Experiments were performed in HeLa cells (ATCC, CRM-CCL-2), FFPE C57BL/6 mouse brain sections (coronal; thickness 5 μm, Acepix Biosciences 7011-0120), FFPE human breast tissue sections (thickness 5 μm; Acepix Biosciences, 7310-0620) and whole-mount zebrafish embryos (wildtype Danio rerio strain AB; fixed at 27 hpf).

Techniques: Imaging, Labeling, Amplification, Multiplexing

Journal: Development (Cambridge, England)

Article Title: Hybridization chain reaction enables a unified approach to multiplexed, quantitative, high-resolution immunohistochemistry and in situ hybridization

doi: 10.1242/dev.199847

Figure Lengend Snippet: Multiplexed protein imaging via HCR 2°IHC using unlabeled primary antibody probes, initiator-labeled secondary antibody probes and simultaneous HCR signal amplification for all targets. (A) Two-stage HCR 2°IHC protocol. Detection stage: unlabeled primary antibody probes bind to protein targets; wash; initiator-labeled secondary antibody probes bind to primary antibody probes; wash. Amplification stage: initiators trigger self-assembly of fluorophore-labeled HCR hairpins into tethered fluorescent amplification polymers; wash. (B) Multiplexing timeline. The same two-stage protocol is used independent of the number of target proteins. (C) Confocal image of 3-plex protein imaging in mammalian cells on a slide; 0.14×0.14 µm pixels; maximum intensity z -projection. Target proteins: PCNA (Alexa647), HSP60 (Alexa546) and SC35 (Alexa488). Sample: HeLa cells. (D) Epifluorescence image of 4-plex protein imaging in FFPE mouse brain sections; 0.6×0.6 µm pixels. Target proteins: TH (Alexa488), GFAP (Alexa546), PVALB (Alexa647) and MBP (Alexa750). (E) Zoom of indicated region in D. Sample: FFPE C57BL/6 mouse brain section (coronal); 5 µm thickness. See sections S5.3 and S5.4 of the supplementary information for additional data.

Article Snippet: Experiments were performed in HeLa cells (ATCC, CRM-CCL-2), FFPE C57BL/6 mouse brain sections (coronal; thickness 5 μm, Acepix Biosciences 7011-0120), FFPE human breast tissue sections (thickness 5 μm; Acepix Biosciences, 7310-0620) and whole-mount zebrafish embryos (wildtype Danio rerio strain AB; fixed at 27 hpf).

Techniques: Imaging, Labeling, Amplification, Multiplexing

Journal: Development (Cambridge, England)

Article Title: Hybridization chain reaction enables a unified approach to multiplexed, quantitative, high-resolution immunohistochemistry and in situ hybridization

doi: 10.1242/dev.199847

Figure Lengend Snippet: qHCR imaging: protein relative quantitation with subcellular resolution in an anatomical context using HCR 1°IHC or HCR 2°IHC. (A) Two-channel redundant detection of a target protein. Top: target protein detected using two primary antibody probes that bind different epitopes, each initiating an orthogonal spectrally distinct HCR amplifier (Ch1, Alexa647; Ch2, Alexa750). Bottom: target protein detected using an unlabeled primary antibody probe and two batches of secondary antibody probes that initiate orthogonal spectrally distinct HCR amplifiers (Ch1, Alexa546; Ch2, Alexa647). (B) Top: epifluorescence image of FFPE mouse brain section; 0.16×0.16 µm pixels. Target protein: TH. Sample: FFPE C57BL/6 mouse brain section (coronal); 5 µm thickness. Bottom: confocal image of FFPE human breast tissue; 0.3×0.3 µm pixels; single optical section. Target protein: KRT17. Sample: FFPE human breast tissue section; 5 µm thickness. (C) High accuracy and precision for protein relative quantitation in an anatomical context. Highly correlated normalized signal (Pearson correlation coefficient, r ) for subcellular voxels in the indicated region in B (top: 2×2 µm voxels in a 5 µm section using epifluorescence microscopy; bottom: 2.0×2.0×2.5 µm voxels using confocal microscopy). Accuracy: linearity with zero intercept. Precision: scatter around the line. See section S5.6 of the supplementary information for additional data.

Article Snippet: Experiments were performed in HeLa cells (ATCC, CRM-CCL-2), FFPE C57BL/6 mouse brain sections (coronal; thickness 5 μm, Acepix Biosciences 7011-0120), FFPE human breast tissue sections (thickness 5 μm; Acepix Biosciences, 7310-0620) and whole-mount zebrafish embryos (wildtype Danio rerio strain AB; fixed at 27 hpf).

Techniques: Imaging, Quantitation Assay, Epifluorescence Microscopy, Confocal Microscopy

Journal: Development (Cambridge, England)

Article Title: Hybridization chain reaction enables a unified approach to multiplexed, quantitative, high-resolution immunohistochemistry and in situ hybridization

doi: 10.1242/dev.199847

Figure Lengend Snippet: Simultaneous multiplexed protein and RNA imaging via HCR 1°IHC + HCR RNA-ISH using initiator-labeled primary antibody probes for protein targets, split-initiator DNA probes for RNA targets, and simultaneous HCR signal amplification for all targets. (A) Three-stage HCR 1°IHC + HCR RNA-ISH protocol. Protein detection stage: initiator-labeled primary antibody probes bind to protein targets; wash. RNA detection stage: split-initiator DNA probes bind to RNA targets; wash. Amplification stage: initiators trigger self-assembly of fluorophore-labeled HCR hairpins into tethered fluorescent amplification polymers; wash. For multiplexed experiments, the same three-stage protocol is used independent of the number of target proteins and RNAs. (B) Confocal image of 4-plex protein and RNA imaging in mammalian cells on a slide; 0.13×0.13 µm pixels; maximum intensity z -projection. Targets: PCNA (protein; Alexa488), HSP60 (protein; Alexa546), U6 (RNA; Alexa594) and ACTB (mRNA; Alexa647). Sample: HeLa cells. (C) Epifluorescence image of 4-plex protein and RNA imaging in FFPE mouse brain sections; 0.16×0.16 µm pixels. Targets: TH (protein; Alexa488), MBP (protein; Alexa546), Prkcd (mRNA; Alexa647) and Slc17a7 (mRNA; Alexa750). Sample: FFPE C57BL/6 mouse brain section (coronal); 5 µm thickness. (D) Zooms of indicated regions in C. See sections S5.7 and S5.8 of the supplementary information for additional data.

Article Snippet: Experiments were performed in HeLa cells (ATCC, CRM-CCL-2), FFPE C57BL/6 mouse brain sections (coronal; thickness 5 μm, Acepix Biosciences 7011-0120), FFPE human breast tissue sections (thickness 5 μm; Acepix Biosciences, 7310-0620) and whole-mount zebrafish embryos (wildtype Danio rerio strain AB; fixed at 27 hpf).

Techniques: Imaging, Labeling, Amplification, RNA Detection

Journal: Development (Cambridge, England)

Article Title: Hybridization chain reaction enables a unified approach to multiplexed, quantitative, high-resolution immunohistochemistry and in situ hybridization

doi: 10.1242/dev.199847

Figure Lengend Snippet: Simultaneous multiplexed protein and RNA imaging via HCR 2°IHC + HCR RNA-ISH using unlabeled primary antibody probes and initiator-labeled secondary antibody probes for protein targets, split-initiator DNA probes for RNA targets, and simultaneous HCR signal amplification for all targets. (A) Three-stage HCR 2°IHC + HCR RNA-ISH protocol. Protein detection stage: unlabeled primary antibody probes bind to protein targets; wash; initiator-labeled secondary antibody probes bind to primary antibody probes; wash. RNA detection stage: split-initiator DNA probes bind to RNA targets; wash. Amplification stage: initiators trigger self-assembly of fluorophore-labeled HCR hairpins into tethered fluorescent amplification polymers; wash. For multiplexed experiments, the same three-stage protocol is used independent of the number of target proteins and RNAs. (B) Confocal image of 4-plex protein and RNA imaging in mammalian cells on a slide; 0.13×0.13 µm pixels; maximum intensity z -projection. Targets: PCNA (protein; Alexa488), HSP60 (protein; Alexa546), U6 (RNA; Alexa594) and HSP60 (mRNA; Alexa647). Sample: HeLa cells. (C) Epifluorescence image of 4-plex protein and RNA imaging in FFPE mouse brain sections; 0.16×0.16 µm pixels. Targets: TH (protein; Alexa488), MBP (protein; Alexa546), Prkcd (mRNA; Alexa647) and Slc17a7 (mRNA; Alexa750). Sample: FFPE C57BL/6 mouse brain section (coronal); 5 µm thickness. (D) Zooms of indicated regions in C. See sections S5.9 and S5.10 of the supplementary information for additional data.

Article Snippet: Experiments were performed in HeLa cells (ATCC, CRM-CCL-2), FFPE C57BL/6 mouse brain sections (coronal; thickness 5 μm, Acepix Biosciences 7011-0120), FFPE human breast tissue sections (thickness 5 μm; Acepix Biosciences, 7310-0620) and whole-mount zebrafish embryos (wildtype Danio rerio strain AB; fixed at 27 hpf).

Techniques: Imaging, Labeling, Amplification, RNA Detection