Journal: Genes & Diseases

Article Title: Rejuvenation of corticospinal neurons enhances rehabilitation-associated corticospinal tract axon sprouting and functional recovery post photothrombotic ischemic stroke in mice

doi: 10.1016/j.gendis.2025.102000

Figure Lengend Snippet: Ectopic expression of OSKTFs in retrogradely labeled corticospinal neurons. (A) Schematic diagrams of the experimental design and the Tet-on system. (B, C) Representative images of transverse brain sections showing retrogradely labeled corticospinal neurons in mice treated with saline or doxycycline (Dox). Scale bar: 100 μm. (D – K) Representative high-magnitude images of layer V sensorimotor cortex showing fluorescent in situ hybridization of tdTomato and transcriptional factors Oct4 , Sox2 , Klf4 , and Fezf2 in mice treated with saline or Dox. Scale bar: 50 μm. (L–O) Quantification of colocalization between tdTomato and Oct4/Klf (L), Sox2 (M), Fezf2 (N), and tdTomato neuronal densities (O) in animals treated with saline ( n = 3) or Dox ( n = 3). ∗∗ P < 0.01; n.s., no statistical significance; Student's t -test. (P, Q) Neonates (P1) or adult (18 weeks) animals received intraspinal AAVRetro-tdTomato or intersectional injection for OSKTFs expression in corticospinal neurons. Mice were euthanized at P7 or 28 weeks with an 8-week Dox or saline treatment (P). Layer V of the sensorimotor cortex was microdissected under an epi-fluorescent microscope (Q) for measuring RNA contents. Scale bar: 500 μm. (R) Relative RNA expression of different histone proteins and epigenetic modulators in P7 mice ( n = 5 biological replicates) and adult mice treated with saline ( n = 5 biological replicates) or Dox ( n = 5 biological replicates). RNA expression levels were first normalized to those of GAPDH and then to those of adult animals treated with saline. ∗∗ P < 0.01; one-way ANOVA followed by the Bonferroni correction.

Article Snippet: To examine ectopic induction of Oct4, Sox2, and Klf4 in adult retrogradely labeled (tdTomato + ) corticospinal neurons, we i) prepared transverse brain sections (15 μm) crossing the RFA and CFA regions in animals with OSKTFs Dox or OSKTFs saline treatment; and ii) performed in situ hybridization using the RNAscope Multiplex Fluorescent Detection Kit v2 (Advanced Cell Diagnostics) following the manufacturer's guidelines.

Techniques: Expressing, Labeling, Saline, In Situ Hybridization, Injection, Microscopy, RNA Expression

Journal: bioRxiv

Article Title: Mitral regurgitation induces a unique fibroblast population associated with atrial fibrillation susceptibility

doi: 10.64898/2026.08.19.745870

Figure Lengend Snippet: RNAscope validation of fibroblast marker expression across MR progression and AF susceptibility. A , Representative 63X RNAscope immunofluorescence images from the six LAPW samples subjected to snRNA-seq. Nuclei are labeled with DAPI (blue). Fibroblast marker transcripts are visualized as ICAM1 (red, Opal 650), GRIA4 (green, Opal 520), ADRB2 (yellow, Opal 570), and PCOLCE2 (white, Opal 780). B , Quantification of total RNAscope puncta counts by condition (n=6 each group). ROIs listed in first panel are representative across each row. C , Quantification by AF inducibility (n=6 each group). D , Quantification by remodeling severity (n=18 total). Individual data represent ROIs, with five ROIs analyzed per tissue section. Pairwise comparisons were performed using Wilcoxon rank-sum tests.

Article Snippet: Spatial validation of candidate fibroblast marker genes identified by snRNA-seq was performed using RNAscope Multiplex Fluorescent In Situ Hybridization (Advanced Cell Diagnostics, Newark, CA, USA) on FFPE left atrial tissue sections .

Techniques: RNAscope, Biomarker Discovery, Marker, Expressing, Immunofluorescence, Labeling

Journal: bioRxiv

Article Title: Mitral regurgitation induces a unique fibroblast population associated with atrial fibrillation susceptibility

doi: 10.64898/2026.08.19.745870

Figure Lengend Snippet: RNAscope validation of fibroblast population marker expression across condition, AF status, and severity. Values are presented as median (IQR) for total RNAscope puncta counts per ROI. Pairwise comparisons were performed using Wilcoxon rank-sum tests. Data are shown for the four fibroblast population markers selected for validation from the snRNA-seq analysis: PCOLCE2, GRIA4, ICAM1, and ADRB2.

Article Snippet: Spatial validation of candidate fibroblast marker genes identified by snRNA-seq was performed using RNAscope Multiplex Fluorescent In Situ Hybridization (Advanced Cell Diagnostics, Newark, CA, USA) on FFPE left atrial tissue sections .

Techniques: RNAscope, Biomarker Discovery, Marker, Expressing

Journal: iScience

Article Title: Astrovirus infection dysregulates goblet cell-associated antigen passages

doi: 10.1016/j.isci.2026.117046

Figure Lengend Snippet:

Article Snippet: In-situ hybridization RNAscope 2.5 HD Assay - RED , Advanced Cell Diagnostics , CAT#322373.

Techniques: Plasmid Preparation, Virus, Expressing, Recombinant, In Situ Hybridization, RNAscope, HD Assay, Enzyme-linked Immunosorbent Assay, Software

Journal: bioRxiv

Article Title: Nociceptor-restricted cannabinoid receptor 1 contributes to chronic but not acute analgesia

doi: 10.64898/2026.08.12.744456

Figure Lengend Snippet: (A) RNAscope i n situ hybridization of Cnr1 (magenta) and Scn10a (green) mRNA in the lumbar (L4-6) DRGs. Scale bar = 20 μm. (B) Average colocalized Cnr1 and Scn10a puncta (Tukey’s multiple comparisons test; n=3-6 animals, * p <0.05 WT vs. CB1R LOXTB ). (C) PCR for Na v 1.8Cre, ZP3Cre, and CB1R-TB. For each gene, a – indicates a WT mice, a -/+ indicates heterozygous Na v 1.8cre/ZP3cre/CB1R-TB, a +/+ indicates homozygous Na v 1.8cre/CB1R-TB. (D) Gene expression revealed insertion of a TB resulted in significant reduction of CB1R gene expression in both male and female lumbar DRGs. Crossing with either Na v 1.8cre (cell specific) or ZP3cre (whole-body) restored CB1R expression to WT levels (Sidak’s multiple comparisons test; n=5 animals, 3 DRGs each, **** p <0.0001 WT vs. CB1R LOXTB ). (E) Cell size distribution of Cnr1 , Scn10a , and colocalized puncta positive cells in WT lumbar DRGs. Cell size distribution of Cnr1 , Scn10a , and colocalized Scn10a puncta positive cells in WT (F), CB1R LOXTB (G), Na v 1.8 +/- :CB1R LOXTB (H), and ZP3 +/- :CB1R LOXTB (I) lumbar DRGs.

Article Snippet: RNA in situ hybridization was performed on fresh frozen DRGs sectioned at 14 μm using the RNAscope in situ hybridization assay (Multiplex Fluorescent Reagent Kit Version 2) with the Advanced Cell Diagnostics (ACD) protocol [ ].

Techniques: RNAscope, Hybridization, Gene Expression, Expressing

Journal: bioRxiv

Article Title: Decorin-mediated Extracellular Matrix Stabilization Maintains Cartilage Integrity During Aging

doi: 10.64898/2026.07.28.741218

Figure Lengend Snippet: a) Decorin immunofluorescence (IF) images illustrate the presence of decorin in articular cartilage from both normal (female, 61 years of age) and osteoarthritis (OA, female 55 years of age) human donors. b) RNAscope in situ hybridization for Dcn mRNA and decorin IF validate decorin ablation in 9-month-old (9M) cartilage-specific inducible Dcn knockout mice ( Dcn flox/flox /AcanCre ER , or cKO ) mice compared to age-matched controls, following the induced ablation at 3 months of age (red: decorin; blue: DAPI, n = 5). c) Representative Safranin-O/Fast Green staining of sagittal knee joint sections at the primary mechanical loading region shows progressive structural disruption and sulfated glycosaminoglycan (sGAG) loss in 9M and 18M cKO cartilage compared to controls ( n ≥ 8). d–f) Quantification of d) cartilage thickness, e) chondrocyte cell density and f) modified Mankin scores from femoral condyle histological sections (mean ± 95% CI; *: p < 0.05; n.s.: not significant; n ≥ 5. Each data point represents the average value measured from one animal). g) High-magnification images of femoral condyle cartilage highlight early surface remodeling at 9M and full erosion of uncalcified cartilage at 18M in cKO joints (black arrowheads). h) Polarized light microscopy (PLM) images of Picrosirius Red (PSR)-stained sections illustrate increased collagen fiber alignment on cKO cartilage surface ( n = 4). i) Scanning electron microscopy (SEM) images show the formation of aligned collagen fibrils along the mediolateral split-line direction (red arrows) on cKO cartilage surface ( n ≥ 5). j–l) Quantitative analysis of collagen fibril organization from SEM images, including j) rose plots of fibril orientation distribution, where 0° corresponds to the mediolateral split-line direction, k) degree of fibril alignment as denoted by the von Mises concentration, κ (mean ± 95% CI), and l) box-and-whisker plot of collagen fibril diameter, d col , distribution ( n = 400 fibrils pooled from n = 4 animals per group). A complete list of quantitative and statistical outcomes is summarized in Supplementary Tables S1 and S2.

Article Snippet: RNAscope in situ hybridization was performed using the RNAscope2.5 HD Detection reagent-RED kit (322350, ACDbio) to assess the spatiotemporal expression of specific murine genes including Col1a1 (319371, ACDbio), Col2a1 (407221, ACDbio), Acan (439101, ACDbio), Dcn (413281, ACDbio), and F5 (502411, ACDbio).

Techniques: Immunofluorescence, RNAscope, In Situ Hybridization, Knock-Out, Staining, Disruption, Modification, Light Microscopy, Electron Microscopy, Concentration Assay, Whisker Assay

Journal: bioRxiv

Article Title: Decorin-mediated Extracellular Matrix Stabilization Maintains Cartilage Integrity During Aging

doi: 10.64898/2026.07.28.741218

Figure Lengend Snippet: a) RNAscope in situ hybridization for Dcn and b) Safranin-O/Fast Green images of sagittal knee joint sections from control (Ctrl) and cKO mice at 9 and 18 months (9M and 18M). Whole-joint views and higher-magnification images of the anterior superior and anterior inferior synovium regions show comparable spatial distribution and intensity of Dcn expression between genotypes. Arrowheads indicate regions of synovial structural alterations. c-e) Histological scores of synovial morphology, including c) cellularity, d) fibrosis, and e) synovial lining hyperplasia (mean ± 95% CI, *: p < 0.05, n.s., not significant; n = 8). Each data point represents the average value measured from one animal.

Article Snippet: RNAscope in situ hybridization was performed using the RNAscope2.5 HD Detection reagent-RED kit (322350, ACDbio) to assess the spatiotemporal expression of specific murine genes including Col1a1 (319371, ACDbio), Col2a1 (407221, ACDbio), Acan (439101, ACDbio), Dcn (413281, ACDbio), and F5 (502411, ACDbio).

Techniques: RNAscope, In Situ Hybridization, Control, Expressing

Journal: bioRxiv

Article Title: Decorin-mediated Extracellular Matrix Stabilization Maintains Cartilage Integrity During Aging

doi: 10.64898/2026.07.28.741218

Figure Lengend Snippet: a) UMAP projections of control (Ctrl) and cKO cartilage cells, after excluding immune and hematopoietic cells. Clusters were annotated based on cluster-enriched marker genes and established lineage markers (see ). Cluster 4 corresponds to articular chondrocytes ( Acan +, Col2a1 +, Col11a2 +, Col1a1– ). b) Dot plot of representative ECM genes and top unique markers for each cluster. Dot size corresponds to the percentage of expressing cells; color denotes average log 2 fold change (log 2 FC ) relative to all other clusters. c) Relative proportion and absolute number of cells per cluster in Ctrl and cKO cartilage. Log 10 fold change (Log 10 FC ) of cell numbers represents differences in cluster abundance between genotypes. d) Left panel, heatmap of selected matrix anabolism, catabolism and TGF-β genes across clusters, row-scaled ( z -score) to highlight relative expression differences between clusters and genotypes. The bar on the right shows relative expression abundance of each gene. Right panel, dot plot of significant differentially expressed genes (DEGs) of cKO versus Ctrl cells (|log 2 FC | > 1, adjusted p < 0.05). e) Volcano plot of significant DEGs in articular chondrocytes (Cluster 4). Dcn is significantly reduced in cKO chondrocytes. f) UMAP feature plots show the expression of representative cartilage ECM and catabolic genes in Ctrl and cKO cells. Cluster 4 cells are highlighted by pink dashed line, reaffirming the targeted reduction of Dcn in cKO articular chondrocytes (pink arrowhead). g) RNAscope in situ hybridization and h) immunofluorescence (IF) images show the spatial distributions of mRNA transcripts and proteins for Acan /aggrecan, Col2a1 /collagen II and Col1a1 /collagen I in femoral condyle cartilage ( n = 4). Insets show the positive controls for RNAscope ( Col2a1 : meniscal horn, Col1a1 : synovium) and internal negative control for IF.

Article Snippet: RNAscope in situ hybridization was performed using the RNAscope2.5 HD Detection reagent-RED kit (322350, ACDbio) to assess the spatiotemporal expression of specific murine genes including Col1a1 (319371, ACDbio), Col2a1 (407221, ACDbio), Acan (439101, ACDbio), Dcn (413281, ACDbio), and F5 (502411, ACDbio).

Techniques: Control, Marker, Expressing, RNAscope, In Situ Hybridization, Immunofluorescence, Negative Control

Journal: bioRxiv

Article Title: Decorin-mediated Extracellular Matrix Stabilization Maintains Cartilage Integrity During Aging

doi: 10.64898/2026.07.28.741218

Figure Lengend Snippet: a) UMAP feature plots showing expression of representative ECM and remodeling genes in control (Ctrl) and cKO cartilage. Cluster 2 migrated cells and Cluster 3 articles chondrocytes are highlighted by dashed circles with colors corresponding to the original UMAPs. b) Dot plot of the top 24 Cluster 2 marker genes identified by FindAllMarkers . Dot size denotes the percentage of expressing cells; color corresponds to average log 2 fold change (log 2 FC ) for each gene within a given cluster relative to all other clusters. c) Gene set enrichment analysis (GSEA) comparing Cluster 2 versus 3 cells. Enriched pathways include ECM remodeling, collagen organization, cell adhesion, and skeletal system development. A complete list of positively and negatively enriched pathways (adjusted p < 0.05) is provided in . d) Top leading-edge genes contributing to enriched pathways associated with ECM remodeling, cell adhesion, and skeletal system development. e) RNAscope in situ hybridization images of Dcn , Acan , and Col1a1 . Positive expression of F5 is observed in cells within the scar-like fibrotic layer, consistent with Cluster 2 transcriptional signatures ( n = 4). f) Immunofluorescence (IF) images of decorin, aggrecan, and collagen I. Insets show internal negative control (dashed white lines highlight cartilage surface and bottom edge of the fibrotic layer in cKO joints, n = 4).

Article Snippet: RNAscope in situ hybridization was performed using the RNAscope2.5 HD Detection reagent-RED kit (322350, ACDbio) to assess the spatiotemporal expression of specific murine genes including Col1a1 (319371, ACDbio), Col2a1 (407221, ACDbio), Acan (439101, ACDbio), Dcn (413281, ACDbio), and F5 (502411, ACDbio).

Techniques: Expressing, Control, Marker, RNAscope, In Situ Hybridization, Immunofluorescence, Negative Control

Journal: Endocrinology

Article Title: Adrenal rather than central dysfunction limits HPA axis recovery after chronic glucocorticoid treatment in male mice

doi: 10.1210/endocr/bqag081

Figure Lengend Snippet: Adrenals were harvested 2 hours after insulin administration (1 u/kg IP). Representative images obtained at 40x magnification from multiplexed fluorescent in situ hybridization (RNAScope, Multiplex Fluorescent V2 Assay, ACDBio) for A) Mc2r (magenta), B) Star , C) Cyp11a1, D Hsd3b2 , and E) Cyp11b1. Data shown as widefield view encompassing all adrenal zones (left, scale bar=100 microns) with magnification of the representative zF region enclosed within the white box (right, scale bar=25 microns) to allow visualization of individual puncta. Quantification of mRNA puncta per zF cell normalized to DN controls: F) Mc2r: p=0.9268 by Kruskal-Wallis test; no significant post-hoc pairwise comparisons vs. DN using Dunn’s correction, G) Star (p=0.056 by one-way ANOVA), H) Cyp11a1 (p=0.44 by Kruskal-Wallis test; no significant post-hoc pairwise comparisons vs. DN), I) Hsd3b2 (p=0.086 by one-way ANOVA; no significant pairwise comparisons vs. DN), J) Cyp11b1 (p=0.0175 by one-way ANOVA). * indicates p<0.05 for post-hoc pairwise comparisons against DN with Bonferroni correction.

Article Snippet: Representative images obtained at 40x magnification from multiplexed fluorescent in situ hybridization (RNAScope, Multiplex Fluorescent V2 Assay, ACDBio) for A) Mc2r (magenta), B) Star , C) Cyp11a1, D Hsd3b2 , and E) Cyp11b1.

Techniques: Gene Expression, In Situ Hybridization, RNAscope, Multiplex Assay

Journal: Endocrinology

Article Title: Adrenal rather than central dysfunction limits HPA axis recovery after chronic glucocorticoid treatment in male mice

doi: 10.1210/endocr/bqag081

Figure Lengend Snippet: WT mice were either dexamethasone unexposed (DN, controls) or treated for 4 days with DEX either as monotherapy (4D DEX) or in combination with cosyntropin (4D DEX+COS). HGRKO mice were treated for 4 days with DEX (HGRKO 4D DEX). Representative images obtained at 40x magnification from fluorescent in situ hybridization (RNAScope, Multiplex Fluorescent V2 Assay, ACDBio) for Mc2r. Data shown as A) widefield views encompassing all adrenal zones (scale bar=100 microns), B) magnification of the boxed zG region, showing grossly similar Mc2r expression in this layer across treatment conditions, and C) magnification of the boxed zF region (scale bar=5 microns for B and C) with D) quantification of Mc2r puncta per zF cell normalized to DN controls (p=0.0263 by one-way ANOVA). *p<0.05 by post-hoc pairwise testing vs. DN with Bonferroni correction.

Article Snippet: Representative images obtained at 40x magnification from multiplexed fluorescent in situ hybridization (RNAScope, Multiplex Fluorescent V2 Assay, ACDBio) for A) Mc2r (magenta), B) Star , C) Cyp11a1, D Hsd3b2 , and E) Cyp11b1.

Techniques: In Situ Hybridization, RNAscope, Multiplex Assay, Expressing