Journal: Cell calcium

Article Title: Injury-induced Erk1/2 signaling tissue-specifically interacts with Ca 2+ activity and is necessary for regeneration of spinal cord and skeletal muscle

doi: 10.1016/j.ceca.2022.102540

Figure Lengend Snippet: Erk1/2 is activated in spinal cord upon injury. Stage-42 larvae were fixed 20 min post-amputation, then processed for transverse sectioning of the tail and co-immunostaining for Sox2 (neural stem cell), Xen1 (pan neuronal membrane) and pErk1/2 (phosphorylated/activated Erk1/2). Non-amputated siblings were fixed, embedded, and sectioned alongside amputated larvae including up to 105 μm posterior to the plane of amputation. A, Representative maximum-intensity projections of tissue sections at the indicated distance anterior to the plane of amputation. Scale bar: 10 μm. B, In approximately every other tissue section, all cells identified by DAPI and within the spinal cord delineated by Xen1 staining were analyzed for Sox2 and pErk1/2 fluorescent signal above an intensity threshold. Sections were pooled by distance from amputation (0 μm) into 50-μm bins: −x posterior (non-amputated only) and + x anterior. Data are mean±SEM percent of Sox2+ (9–42 cells/section except for amputated samples within 112 μm of the amputation: 0–24 cells/section) and Sox2− (8–44 cells/section) cells that are pErk1/2+. N ≥ 3 independent experiments for total n = 4–6 larvae per treatment group. Difference in% pErk1/2+ between amputated and non-amputated larvae was analyzed by ANOVA by bin, and color-coded stars indicate * p < 0.05, ** p < 0.001 in Sox2+ (dark blue) or Sox2− (light blue) cells.

Article Snippet: Slides were probed with antibodies against SOX2 1:100 (R&D AF2018), pERK1/2 1:50 (Cell Signaling 4377 s), and Xen1 1:100 (deposited to Developmental Studies Hybridoma Bank (DSHB) by Ruiz i Altaba, A., AB 531,871) overnight at 4 °C.

Techniques: Immunostaining, Staining

Journal: bioRxiv

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

doi: 10.1101/2021.06.02.446311

Figure Lengend Snippet: (A) 2-stage 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 2-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 µ m pixels; maximum intensity z-projection. Target proteins: HSP60 (Alexa488), GM130 (Alexa647), SC35 (Alexa546). Sample: HeLa cells. (D) Epifluorescence image of 4-plex protein imaging in FFPE mouse brain sections; 0.3 × 0.3 µ m pixels. Target proteins: TH (Alexa488), GFAP (Alexa546), MBP (Alexa647), MAP2 (Alexa750). (E) Zoom of depicted region of panel D. Sample: FFPE C57BL/6 mouse brain section (coronal); thickness: 5 µ m. See Section S5.2 for additional data.

Article Snippet: Experiments were performed in HeLa cells (ATCC Cat. # CRM-CCL-2), human embryonic kidney (HEK) 293T cells (ATCC Cat. # CRL-3216), FFPE C57BL/6 mouse brain sections (coronal; thickness 5 µ m, Acepix Biosciences Cat. # 7011-0120), FFPE human breast tissue sections (thickness 5 µ m; Acepix Biosciences Cat. # 7310-0620), and whole-mount zebrafish embryos (fixed 27 hpf).

Techniques: Labeling, Amplification, Multiplexing, Imaging

Journal: bioRxiv

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

doi: 10.1101/2021.06.02.446311

Figure Lengend Snippet: (A) 2-stage 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 2-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 µ m pixels; maximum intensity z-projection. Target proteins: PCNA (Alexa647), HSP60 (Alexa546), SC35 (Alexa488). Sample: HeLa cells. (D) Epifluorescence image of 4-plex protein imaging in FFPE mouse brain sections; 0.6 0.6 µ m pixels. Target proteins: TH (Alexa488), GFAP (Alexa546), PVALB (Alexa647), MBP (Alexa750). (E) Zoom of depicted region of panel D. Sample: FFPE C57BL/6 mouse brain section (coronal); thickness: 5 µ m. See Section S5.3 for additional data.

Article Snippet: Experiments were performed in HeLa cells (ATCC Cat. # CRM-CCL-2), human embryonic kidney (HEK) 293T cells (ATCC Cat. # CRL-3216), FFPE C57BL/6 mouse brain sections (coronal; thickness 5 µ m, Acepix Biosciences Cat. # 7011-0120), FFPE human breast tissue sections (thickness 5 µ m; Acepix Biosciences Cat. # 7310-0620), and whole-mount zebrafish embryos (fixed 27 hpf).

Techniques: Labeling, Amplification, Multiplexing, Imaging

Journal: bioRxiv

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

doi: 10.1101/2021.06.02.446311

Figure Lengend Snippet: (A) 2-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 unlabeled primary antibody probes 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 µ m pixels. Target protein: TH. Sample: FFPE C57BL/6 mouse brain section (coronal); thickness: 5 µ m. Bottom: Confocal image of FFPE human breast tissue; 0.3 × 0.3 µ m pixels; single optical section. Target protein: KRT17. Sample: FFPE human breast tissue section; thickness: 5 µ m. (C) High accuracy and precision for protein relative quantitation in an anatomical context. Highly correlated normalized signal (Pearson correlation coefficient, r ) for subcellular voxels (2 × 2 µ m) in the depicted region of panel B. Accuracy: linearity with zero intercept. Precision: scatter around the line. See Section S5.6 for additional data.

Article Snippet: Experiments were performed in HeLa cells (ATCC Cat. # CRM-CCL-2), human embryonic kidney (HEK) 293T cells (ATCC Cat. # CRL-3216), FFPE C57BL/6 mouse brain sections (coronal; thickness 5 µ m, Acepix Biosciences Cat. # 7011-0120), FFPE human breast tissue sections (thickness 5 µ m; Acepix Biosciences Cat. # 7310-0620), and whole-mount zebrafish embryos (fixed 27 hpf).

Techniques: Quantitation Assay

Journal: bioRxiv

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

doi: 10.1101/2021.06.02.446311

Figure Lengend Snippet: (A) 3-stage IHC + 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 3-stage protocol is used independent of the number of target proteins and RNAs. (B) Confocal image of 4-plex IHC + RNA-ISH in mammalian cells on a slide; 0.13 × 0.13 µ m pixels; maximum intensity z-projection. Targets: PCNA (protein; Alexa488), HSP60 (protein; Alexa546), U6 (RNA,; Alexa594), ACTB (mRNA; Alexa647). Sample: HeLa cells. (C) Confocal image of 4-plex IHC + RNA-ISH in FFPE mouse brain sections; 0.16 × 0.16 µ m pixels. Targets: TH (protein; Alexa488), MBP (protein; Alexa546), Prkcd (mRNA; Alexa647), Slc17a7 (mRNA; Alexa750). Sample: FFPE C57BL/6 mouse brain section (coronal); thickness: 5 µ m. (D) Zoom of depicted regions of panel C. See Section S5.7 for additional data.

Article Snippet: Experiments were performed in HeLa cells (ATCC Cat. # CRM-CCL-2), human embryonic kidney (HEK) 293T cells (ATCC Cat. # CRL-3216), FFPE C57BL/6 mouse brain sections (coronal; thickness 5 µ m, Acepix Biosciences Cat. # 7011-0120), FFPE human breast tissue sections (thickness 5 µ m; Acepix Biosciences Cat. # 7310-0620), and whole-mount zebrafish embryos (fixed 27 hpf).

Techniques: Labeling, RNA Detection, Amplification

Journal: bioRxiv

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

doi: 10.1101/2021.06.02.446311

Figure Lengend Snippet: (A) 3-stage IHC + 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 3-stage protocol is used independent of the number of target proteins and RNAs. (B) Confocal image of 4-plex IHC + RNA-ISH in mammalian cells on a slide; 0.13 × 0.13 µ m pixels; maximum intensity z-projection. Targets: PCNA (protein; Alexa488), HSP60 (protein; Alexa546), U6 (RNA,; Alexa594), HSP60 (mRNA; Alexa647). Sample: HeLa cells. (C) Confocal image of 4-plex IHC + RNA-ISH in FFPE mouse brain sections; 0.16 × 0.16 µ m pixels. Targets: TH (protein; Alexa488), MBP (protein; Alexa546), Prkcd (mRNA; Alexa647), Slc17a7 (mRNA; Alexa750). Sample: FFPE C57BL/6 mouse brain section (coronal); thickness: 5 µ m. (D) Zoom of depicted regions of panel C. See Section S5.8 for additional data.

Article Snippet: Experiments were performed in HeLa cells (ATCC Cat. # CRM-CCL-2), human embryonic kidney (HEK) 293T cells (ATCC Cat. # CRL-3216), FFPE C57BL/6 mouse brain sections (coronal; thickness 5 µ m, Acepix Biosciences Cat. # 7011-0120), FFPE human breast tissue sections (thickness 5 µ m; Acepix Biosciences Cat. # 7310-0620), and whole-mount zebrafish embryos (fixed 27 hpf).

Techniques: Labeling, RNA Detection, Amplification