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: Communications Biology

Article Title: Targeted volumetric single-molecule localization microscopy of defined presynaptic structures in brain sections

doi: 10.1038/s42003-021-01939-z

Figure Lengend Snippet: a View of a virtual mouse brain with the dimensions of the hippocampal formation in green and a horizontal section from Allen Mouse Brain Atlas (image credit: Allen Institute for Brain Science, available from: http://mouse.brain-map.org/static/brainexplorer ) to demonstrate the hippocampal region used for imaging (white box). b Confocal image of mossy fibers in a 25 µm thick hippocampal slice from a Thy1mEGFP(Lsi1) mouse. The white box marks again the region of interest (25 µm × 25 µm × 25 µm) that is shown magnified in the next panel. c 3D volume reconstruction of mEGFP in xyz -view, showing axons and mossy fiber boutons (green), a typical bouton with filopodial extensions is marked and further highlighted in d . The window (10 µm in z ) for en bloc imaging in the 25 µm thick tissue block is shown in blue. d 3D volume reconstruction of a mossy fiber bouton. The light green area of the bouton shows the volume that would be captured using a typical 1 µm imaging depth in z for a single focal plane. The white box marks a virtual synaptic contact that is further magnified in the next panel. e A virtual synaptic contact in side view (presynaptic membrane in green, Bassoon clusters in magenta, Homer-1 in yellow, postsynaptic membrane in gray), scale bar in b 200 µm, in e 100 nm.

Article Snippet: Fig. 2 Two-color imaging of hippocampal mossy fibers and Bassoon clusters along the ventro-dorsal axis. a Ventral view of a virtual mouse brain with landmarks for trimming in yellow (left) and horizontal sections from Allen Mouse Brain Atlas (image credit: Allen Institute for Brain Science, available from: https://mouse.brain-map.org/static/brainexplorer ) to illustrate the ventral (level 1, middle panel) and the 600 μm more dorsal region (level 2). b Confocal image of mossy fibers in a Thy1EGFP(M) mouse with zinc-transporter 3 (magenta) staining.

Techniques: Imaging, Blocking Assay, Membrane

Journal: Communications Biology

Article Title: Targeted volumetric single-molecule localization microscopy of defined presynaptic structures in brain sections

doi: 10.1038/s42003-021-01939-z

Figure Lengend Snippet: a Ventral view of a virtual mouse brain with landmarks for trimming in yellow (left) and horizontal sections from Allen Mouse Brain Atlas (image credit: Allen Institute for Brain Science, available from: https://mouse.brain-map.org/static/brainexplorer ) to illustrate the ventral (level 1, middle panel) and the 600 µm more dorsal region (level 2). b Confocal image of mossy fibers in a Thy1EGFP(M) mouse with zinc-transporter 3 (magenta) staining. A white box marks the imaging window (30 µm × 30 µm) shown enlarged in the next panel. c Two-color d STORM image of synapses stained for presynaptic Bassoon (green) and postsynaptic Homer 1 (magenta). White box indicates magnified view in the next panel. d , e Synaptic contacts in different orientations: upper box—side view, lower box—plane view further magnified in e . f Protein domain layout of Mus musculus Bassoon and Homer1: Zinc-finger (Zn), coiled coil (CC), PxxP, and enabled/VASP homology (EVH); epitopes of the anti-Bassoon (green) and anti-Homer 1 (magenta) antibodies. Histograms of length ( g ) and counts ( i ) of Bassoon clusters in side view at level 1 (blue, n = 181 clusters in nine images from eight animals) and level 2 (magenta, n = 208 clusters in eight images from eight animals). Clusters at level 2 are longer ( p < 0.001) and have more counts ( p < 0.05) than at level 1. Summary box plots (horizontal line median, boxes 25th and 75th, whiskers 10th and 90th percentile) in all eight individual animals for length ( h ) and localization counts ( j ) (* p < 0.05; ** p < 0.01; *** p < 0.001). i Scale bars 100 µm ( b ), 3 µm ( c ), 1 µm ( d ), and 200 nm ( e ).

Article Snippet: Fig. 2 Two-color imaging of hippocampal mossy fibers and Bassoon clusters along the ventro-dorsal axis. a Ventral view of a virtual mouse brain with landmarks for trimming in yellow (left) and horizontal sections from Allen Mouse Brain Atlas (image credit: Allen Institute for Brain Science, available from: https://mouse.brain-map.org/static/brainexplorer ) to illustrate the ventral (level 1, middle panel) and the 600 μm more dorsal region (level 2). b Confocal image of mossy fibers in a Thy1EGFP(M) mouse with zinc-transporter 3 (magenta) staining.

Techniques: Staining, Imaging