Journal: The American Journal of Pathology

Article Title: Progression and Resolution of SARS-CoV-2 Infection in Golden Syrian Hamsters

doi: 10.1016/j.ajpath.2021.10.009

Figure Lengend Snippet: In situ hybridization and immunohistochemistry findings in lung. RNAscope in situ hybridization showed widespread, punctate staining in the lungs 2 and 4 DPI, but was present only in a small focus within one animal at 7 DPI. No staining was present at 14 DPI (RNAscope in situ hybridization with red chromogen and hematoxylin counterstain). Immunostaining to detect Iba-1 revealed that macrophages comprised a major portion of the inflammatory infiltrate beginning at 2 DPI and peaking at 7 DPI. CD3+ lymphocytes represented a much smaller portion of the inflammatory population in the lungs but also increased from 2-7 DPI then decreased at 14 DPI. (Immunostaining with brown chromogen and hematoxylin counterstain). 20x magnification.

Article Snippet: 10% NBF-fixed lung sections of SARS-CoV-2 infected and control animals were immunostained with anti-Iba-1 antibody (1:1000; Wako; 019-19741 Richmond, VA), anti-CD3 antibody (1:200; DAKO; Ref: A0452 Santa Clara, CA, or anti-pan-cytokeratin antibody (1:1000; Santa Cruz; sc-8018 Dallas, TX).

Techniques: In Situ Hybridization, Immunohistochemistry, RNAscope, Staining, Immunostaining

Journal: The American Journal of Pathology

Article Title: Progression and Resolution of SARS-CoV-2 Infection in Golden Syrian Hamsters

doi: 10.1016/j.ajpath.2021.10.009

Figure Lengend Snippet: Quantitative digital image analysis of ISH and IHC staining in the lungs using QuPath analysis. A. Quantitation of staining for SARS-CoV-2 RNA within the lungs showed highest levels at 2 DPI, with virus still detectable at 4 DPI. No measurable virus was detected at 7 or 14 DPI. B. Copy numbers of viral RNA within the lungs detected by qRT-PCR reached highest levels at 2 and 4 DPI, with progressively lower amounts detected at 7 DPI extending through 28 DPI. C. Quantitation of Iba-1 immunolabeling within the lungs revealed an increase in macrophages in infected animals until 7 DPI, with a decrease at 14 DPI, though still above levels found in mock-inoculated animals. D. Quantitation of CD3+ immunolabeling within the lungs revealed an increase in lymphocytes until 4-7 DPI. Numbers of lymphocytes were 10-fold lower than macrophages. ***p<0.001, ****p<0.0001; region of interest (ROI) was entire left lung lobe.

Article Snippet: 10% NBF-fixed lung sections of SARS-CoV-2 infected and control animals were immunostained with anti-Iba-1 antibody (1:1000; Wako; 019-19741 Richmond, VA), anti-CD3 antibody (1:200; DAKO; Ref: A0452 Santa Clara, CA, or anti-pan-cytokeratin antibody (1:1000; Santa Cruz; sc-8018 Dallas, TX).

Techniques: Immunohistochemistry, Quantitation Assay, Staining, Virus, Quantitative RT-PCR, Immunolabeling, Infection

Journal: Nucleic acids research

Article Title: Digital direct detection of microRNAs using single molecule arrays.

doi: 10.1093/nar/gkx542

Figure Lengend Snippet: Figure 1. Schematic of miRNA detection using Simoa. (A) Overview of the sandwich protocol. Capture probes were covalently coupled to microbeads, and then incubated with target miRNA and biotinylated detection probes to form a sandwich complex. The beads were washed and incubated with SG and RGP (enzyme and substrate) to produce a fluorescent product. (B) Detection was performed in the Simoa format as previously reported (32). As shown in the side view, following hybridization, microbeads suspended in fluorogenic substrate were loaded onto an array of femtoliter-size wells. After loading, the wells were sealed with oil, resulting in an array of isolated reaction chambers each of which contained either zero or one bead. If the enzyme-labeled complex was present on a bead, it generated a fluorescent product resulting in a detectable fluorescent signal. The array was imaged and analyzed to determine the total number of beads, and the number of ‘active’ wells was counted to calculate the average enzyme per bead (AEB). As shown in the top view, the number of active wells increased with increasing target concentration.

Article Snippet: To determine concentrations of miRNAs in these samples, a calibration curve for each target miRNA was fit to a 4PL nonlinear regression with a 1/y2 weighting factor, and unknown values were interpolated using the Simoa HD-1 Analyzer Software (Quanterix).

Techniques: Incubation, Hybridization, Isolation, Labeling, Generated, Concentration Assay

Journal: Nucleic acids research

Article Title: Digital direct detection of microRNAs using single molecule arrays.

doi: 10.1093/nar/gkx542

Figure Lengend Snippet: Figure 3. Three-plex Simoa assay for miR-21, miR-141 and miR-16, and cross reactivity profiles. (A) Multiplex assay for the simultaneous direct detection of three different miRNAs. (B–D) Cross-reactivity of the multiplex assay in the presence of (B) only miR-21, (C) only miR-141 and (D) only miR-16.

Article Snippet: To determine concentrations of miRNAs in these samples, a calibration curve for each target miRNA was fit to a 4PL nonlinear regression with a 1/y2 weighting factor, and unknown values were interpolated using the Simoa HD-1 Analyzer Software (Quanterix).

Techniques: Multiplex Assay

Journal: Nucleic acids research

Article Title: Digital direct detection of microRNAs using single molecule arrays.

doi: 10.1093/nar/gkx542

Figure Lengend Snippet: Figure 4. Three-plex Simoa assay for let-7a, let-7b and let-7c and cross-reactivity profiles. (A) Target, capture, and detection probe sequences used in this assay, and multiplex assay results for the direct detection of three different miRNAs simultaneously. (B–D) Cross-reactivity of the multiplex assay in the presence of (B) only let-7a, (C) only let-7b and (D) only let-7c. (E) Detection of let-7a, let-7b and let-7c in 12 samples using the three-plex Simoa assay. Actual spike-in concentrations for each of let-7a, let-7b and let-7c in the samples (S1–S12) are given in the table, and measured concentrations (after compensating for off-target signal as shown in Supplementary Figure S3 and Supplementary Table S3) are indicated in the plot.

Article Snippet: To determine concentrations of miRNAs in these samples, a calibration curve for each target miRNA was fit to a 4PL nonlinear regression with a 1/y2 weighting factor, and unknown values were interpolated using the Simoa HD-1 Analyzer Software (Quanterix).

Techniques: Multiplex Assay

Journal: Nucleic acids research

Article Title: Digital direct detection of microRNAs using single molecule arrays.

doi: 10.1093/nar/gkx542

Figure Lengend Snippet: Figure 6. Direct detection of miR-21, miR-141 and miR-16 in a total RNA sample. Samples were serially diluted and concentrations were measured using the three-plex Simoa assay. (A) AEB values of the samples at varying amounts of total RNA. (B) Concentrations of miR-21, miR-141 and miR-16 in varying amounts of total RNA measured using RT-qPCR and the three-plex Simoa assay.

Article Snippet: To determine concentrations of miRNAs in these samples, a calibration curve for each target miRNA was fit to a 4PL nonlinear regression with a 1/y2 weighting factor, and unknown values were interpolated using the Simoa HD-1 Analyzer Software (Quanterix).

Techniques: Quantitative RT-PCR