grna fish probes  (Proteintech)

Journal: bioRxiv

Article Title: Multi-color super-resolution imaging to study human coronavirus RNA during cellular infection

doi: 10.1101/2021.06.09.447760

Figure Lengend Snippet: (A) Top: 229E genomic construct map used for the detection of viral genome in fixed cells. 48 FISH probes were designed to target genomic RNA (gRNA) by hybridizing with the RNA-dependent RNA polymerase (RdRp)-coding region, which is only present in the positive-sense gRNA and not in the subgenomic RNAs (sgRNAs). Bottom: Viral genome compared to sgRNAs, which do not contain complementary sequences to the FISH probes. The FISH probes are labeled by CF568 or AF647. Adapted from templates “Discontinuous Transcription” and “Remdesivir Active Molecule Interaction with SARS-CoV-2 RdRp”, by BioRender.com (2021). Retrieved from https://app.biorender.com/biorender-templates . ( Hartenian et al ., 2020 ) (B) Scheme showing double-stranded RNA (dsRNA) targeted by anti-dsRNA antibodies in fixed cells. Each anti-dsRNA antibody recognizes 40 bp of dsRNA. A CF568-labeled secondary antibody is then introduced for visualization. (C) Scheme showing the endoplasmic reticulum (ER) visualized in fixed cells via the expression of a single-pass ER membrane protein, GFP-Sec61B. An AF647-labeled anti-GFP nanobody is used to perform super-resolution (SR) imaging of the ER. (D) Representative confocal images of gRNA (magenta) in GFP-Sec61B-expressing fixed MRC5 cells (gray). Cells were infected with 0.2 MOI 229E and fixed at 6-, 12-, and 24-hour post infection (h p.i.). Nucleus is labeled with DAPI (blue). Scale bar: 10 μm. Right: insets of dotted boxes. Scale bar: 5 μm. (E) Representative confocal images of dsRNA (green) in GFP-Sec61B-expressing fixed MRC5 cells (gray) at 6, 12, and 24 h p.i. with 0.2 MOI 229E. The nucleus is labeled with DAPI (blue). Scale bar: 10 μm. Right: insets of dotted boxes. Scale bar: 5 μm.

Article Snippet: For simultaneous staining of gRNA and ER membrane, transduced cells were incubated with 100 μL Hybridization Buffer containing 2 μL 12.5 μM CF568-labled gRNA FISH probes and 1:2000 AF647-labeled anti-GFP nanobody (Chromotek, gb2AF647–50) for 4 hours in the dark.

Techniques: Construct, Labeling, Expressing, Imaging, Infection

Journal: bioRxiv

Article Title: Multi-color super-resolution imaging to study human coronavirus RNA during cellular infection

doi: 10.1101/2021.06.09.447760

Figure Lengend Snippet: (A) Spatial distribution of fluorescently labeled gRNA in an infected cell via SR microscopy. Numerous nanoscale gRNA puncta, presumably packaged virions, appeared sometimes near the extended clusters. (B) SR reconstruction of isolated virions plated on a coverslip, extracted from cells labeled with the same FISH protocol employed for cellular imaging. Their appearance is very similar to the observed nanoscale gRNA puncta in the cell. Scale bar: 500 nm. (C) Size distribution of purified virions, calculated by fitting the virions with a 2D Gaussian. The mean full width half max of the purified virions was 71 nm. N = 525. (D) DL images of purified virions, immobilized on glass coverslips. FISH probes specifically targeted the viral genome with minimal nonspecific background, as evident from the control condition without virions. Scale bar: 2 μm. (E) Number of FISH probes per virion, calculated by dividing the total fluorescence intensity of individual virions by the single-molecule brightness. On average, each virion is labeled by 18.5 FISH probes. N=441. (F) Plot of virion sizes versus brightness, colored according to the normalized local density of points. There is no clear correlation between the two parameters (Pearson correlation = 0.28). (G) SR reconstructions of nine unique virions stained with spike protein antibodies. The virions are 100–150 nm diameter large bright objects. Scale bar: 100 nm. (H) DL images of two different virions labeled with spike protein (green) and gRNA (magenta). The white color indicates clear colocalization in the center. (I) Two-color SR reconstructions of virions shown in panel H. A distinctive concentric structure of gRNA encapsulated by a larger structure labeled with the spike protein is clearly observed (J) Additional two-color SR reconstructions of several virions where colocalization is evident. 39% of the total virions observed (N=168) exhibited some degree of colocalization. Scale bar: 100 nm. (K) Size distributions of purified virions stained with spike protein antibody. The mean full width half maximum is 119 nm. N=26. (L) Percentage area distribution of gRNA clusters for cells 6 (black), 12 (magenta) and 24 (turquoise) h p.i. Cells infected for a longer period of time showed larger clusters representing increasing copy number of the viral genome. Data collected from 9, 10 and 8 cells, respectively. (M) Cluster density for cells 6 (black), 12 (magenta) and 24 (turquoise) h p.i. Cells infected for a longer period of time exhibited a higher cluster density. *, p< 10 −1 ; **, p< 10 −2 (two-tailed t-test). Data collected from 9, 10 and 8 cells, respectively.

Article Snippet: For simultaneous staining of gRNA and ER membrane, transduced cells were incubated with 100 μL Hybridization Buffer containing 2 μL 12.5 μM CF568-labled gRNA FISH probes and 1:2000 AF647-labeled anti-GFP nanobody (Chromotek, gb2AF647–50) for 4 hours in the dark.

Techniques: Labeling, Infection, Microscopy, Isolation, Imaging, Purification, Fluorescence, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: Multi-color super-resolution imaging to study human coronavirus RNA during cellular infection

doi: 10.1101/2021.06.09.447760

Figure Lengend Snippet: (A) Representative confocal images of HCoV-229E-infected MRC5 cells in control and Remdesivir (0.1 μM and 0.5 μM) treated cells using the same brightness and contrast threshold, labeled with FISH probes targeting gRNA (magenta), anti-dsRNA antibody (green) and nuclear staining (blue). Scale bar: 50 μm. (B) Representative confocal images showing gRNA (magenta) and dsRNA (green) in control and 0.1μM Remdesivir-treated MRC5 cells at 24 h p.i. Right: Quantification of the number of dsRNA puncta per cell in control and 0.1μM Remdesivir-treated (RDV) MRC5 cells at 24 h p.i. Scale bar: 10 μm. ****, p< 10 −4 . Data collected from 21 and 25 cells, respectively. (C-D) DL images and corresponding SR reconstructions of two regions of a cell incubated with 0.1 μM Remdesivir at 24 h p.i. where gRNA (magenta) and dsRNA (green) are labeled. dsRNA puncta appeared at the periphery of gRNA clusters, again anticorrelated. (E) Spatial point statistics verify anticorrelation of gRNA and dsRNA. CSR is simulated with the same signal density (black). (F) Sizes of gRNA clusters for Remdesivir-treated cells and untreated cells. Remdesivir treatment reduced the size of the gRNA clusters. *, p< 10 −1 (two-tailed t-test). (G) Sizes of dsRNA puncta for drug-treated cells and untreated cells. **, p< 10 −2 (two-tailed t-test).

Article Snippet: For simultaneous staining of gRNA and ER membrane, transduced cells were incubated with 100 μL Hybridization Buffer containing 2 μL 12.5 μM CF568-labled gRNA FISH probes and 1:2000 AF647-labeled anti-GFP nanobody (Chromotek, gb2AF647–50) for 4 hours in the dark.

Techniques: Infection, Labeling, Staining, Incubation, Two Tailed Test