rnascope 4-plex kit reagents  (Advanced Cell Diagnostics Inc)

Journal: eLife

Article Title: Spatially resolved transcriptomics reveals pro-inflammatory fibroblast involved in lymphocyte recruitment through CXCL8 and CXCL10

doi: 10.7554/eLife.81525

Figure Lengend Snippet: ( A ) Single-cell RNA sequencing integration with spatial data using ‘anchor’-based integration workflow in Seurat to enable a prediction score for each spot for fibroblast 5. Based on this prediction score, fibroblast 5 is confirmed spatially restricted. ( B ) Multiplex mRNA in situ hybridisation of a representative section of human oral mucosa using specific probes against RAC2 and LCP1 (fibroblast 5 markers), CD14 (macrophages), and CD79A (B cells) to validate in vivo localisation of fibroblast 5 and immune cell populations. Scale bars: 250 µm. ( C ) Schematic illustration of our proposed model summarising pathogenic fibroblast population role in human oral chronic disease. Created with Biorender.

Article Snippet: Multiplex ISH was performed using kits and probes from Advanced Cell Diagnostics (ACD) which used oligonucleotide probes to RNA targets (RNAscope 4-plex Ancillary Kit for Multiplex Fluorescent Reagent Kit v2; Cat# 323120) as per the manufacturer’s instructions with incubations steps of processed paraffin-embedded tissue performed in HybEz oven (ACD, Cat# 321461).

Techniques: RNA Sequencing, Multiplex Assay, In Situ, Hybridization, In Vivo

Journal: Cell Reports

Article Title: Resolving the immune landscape of human prostate at a single-cell level in health and cancer

doi: 10.1016/j.celrep.2021.110132

Figure Lengend Snippet:

Article Snippet: RNAscope® LS 4-Plex Ancillary Kit Multiplex Reagent Kit , Advanced Cell Diagnostics , Cat#322830.

Techniques: Control, Zinc Assay, Multiplex Assay, RNAscope, Gene Expression, Software

Journal: Cell Reports

Article Title: Resolving the immune landscape of human prostate at a single-cell level in health and cancer

doi: 10.1016/j.celrep.2021.110132

Figure Lengend Snippet: Immune landscape of the prostate includes a prostate-specific macrophage subset enriched in metallothionein transcripts (A) UMAP of 793 cells in myeloid compartment after integration of myeloid/MNP cells from n = 10 patients with Henry et al. myeloid/MNP cells. (B) Mean expression dot plot of top five significant marker genes for each myeloid cluster. Marker genes were identified using Wilcoxon rank sum test and p adj < 0.05 was considered statistically significant. Size of circles indicate percentage of cells expressing the gene and increasing color gradient from white to red corresponds to increasing expression value. (C) UMAP plot of predicted MNP clusters in prostate cancer single cell data from ( Karthaus et al., 2020 , Chen et al., 2021 , Crowley et al., 2020 ). (D) (top) Representative RNAscope images of probes targeting MT1 family genes (magenta) and CD68 (yellow). ‘L’ indicates lumen. Arrows point to single cells that are marked by both probes in sub-panels i and ii. Scale bar, 20 μm. (bottom) Representative immunofluorescence microscopy images of a human prostate section labeled for metallothionein (α-MT)/isotype control (yellow), HLA-DR (cyan), CD206 (purple) and DAPI (blue). White arrows point to structure displaying colocalization of α-MT with HLA-DR and/or CD206 labeling. Scale bars, 50 μm. (E) Mean expression dot plot of Zinc transporter genes for each myeloid cluster. Size of circles indicate percentage of cells expressing the gene and increasing color gradient from white to red corresponds to increasing expression value. (F) Heatmap of mean AUCell enrichment of F4/80 hi/lo gene sets, corresponding to yolk sac (YS) versus hematopoetic stem-cell (HSC) lineage. Row enrichment value is scaled from 0 to 1 and presented as an increasing gradient from black, gray, yellow to orange which corresponds to increasing enrichment score. (G) Representative immunofluorescence microscopy images of cross sections of mouse prostate labeled for F4/80 (green), MHCII (red), CD11b (blue), CD31 (yellow) and phalloidin. Scale bars, 120 μm. (H) Cell counts per gram of prostate for rat IgG2a isotype or anti-Csf1r antibody (Ab) treated male mice. N = 5 per group. ∗∗∗∗ p < 0.0001; n.s denotes not significant (p > 0.05) (Two-way ANOVA with Tukey’s multiple correction). (I) Zinc concentration of anterior prostate lobe, liver lobe, and kidney from male mice treated with either rat IgG2a isotype control or anti-Csf1r Ab. N = 6 per group. (shown is representative quantification from one of two independent experiments). ∗ p < 0.05; n.s. not significant (Mann-Whitney test). See also .

Article Snippet: Simultaneous detection of human CD68 and MT1 family genes were performed on FFPE sections using Advanced Cell Diagnostics (ACD) RNAscope® 2.5 LS Multiplex Reagent Kit (Cat No. 322800), RNAscope® LS 4-Plex Ancillary Kit Multiplex Reagent Kit (Cat No. 322830), RNAscope® 2.5 LS Probes (ACD, Hayward, CA, USA) at the histopathology/ in situ hybridization core facility at Cancer Research UK – Cambridge Institute.

Techniques: Expressing, Marker, Immunofluorescence, Microscopy, Labeling, Concentration Assay, MANN-WHITNEY

Journal: Cell Reports

Article Title: Resolving the immune landscape of human prostate at a single-cell level in health and cancer

doi: 10.1016/j.celrep.2021.110132

Figure Lengend Snippet:

Article Snippet: Simultaneous detection of human CD68 and MT1 family genes were performed on FFPE sections using Advanced Cell Diagnostics (ACD) RNAscope® 2.5 LS Multiplex Reagent Kit (Cat No. 322800), RNAscope® LS 4-Plex Ancillary Kit Multiplex Reagent Kit (Cat No. 322830), RNAscope® 2.5 LS Probes (ACD, Hayward, CA, USA) at the histopathology/ in situ hybridization core facility at Cancer Research UK – Cambridge Institute.

Techniques: Zinc Assay, Multiplex Assay, Expressing, Software