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: 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: 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