rnascope acdbio multiplex v2  (Advanced Cell Diagnostics Inc)

Journal: Advanced Science

Article Title: Morphine Tolerance Gated through EZH2‐Mediated Suppression of Trpc5 in Spinal GABAergic Interneurons in Male Mice

doi: 10.1002/advs.202507908

Figure Lengend Snippet: Morphine downregulates TRPC5 in the lamina I‐III of spinal dorsal horn. A) KEGG pathway analysis displayed that the most significantly enriched signaling pathways of differential expression genes in the spinal cords between morphine‐treated group and saline‐treated group. B) Volcano plot showed the results of differential expression genes analysis performed in morphine‐treated group relative to saline‐treated group. Genes related with morphine tolerance and pain are indicated. C) The heatmap showed mRNA expression in the spinal cords from mice after morphine treatment and saline treatment based on RNA sequence ( n = 3 samples/group, each sample included 3 L4‐L5 spinal cords). Colors represent high (red) and low (blue) intensity. D) In situ hybridization RNAscope images of Trpc5 mRNA in the spinal dorsal horn of saline‐treated group and morphine‐treated mice. White arrows indicated Trpc5 mRNA puncta. E) Quantitative analysis showed decreased Trpc5 + puncta per cell in the spinal dorsal horn after chronic morphine exposure compared to the saline group (Unpaired Student's t test, t (6) = 8.06, P = 0.0002, n = 4). F) Representative images of immunohistochemistry showed the downregulated protein level of TRPC5 in the spinal dorsal horn from morphine‐treated group compared with saline‐treated group. White arrows indicated TRPC5. n = 3. G) Immunoblotting data showed the levels of TRPC5 expression in L4‐L5 sections of the spinal cords after morphine exposure (i.t., 10 µg/10 µL) from day 0 to day 7 (one‐way ANOVA, F (7,16) = 8.250, P < 0.0001, n = 3). H) Representative confocal microscopy images of TRPC5 in the dorsal horn lamina I‐III of mice with neurons (NeuN), astrocytes (GFAP) and microglia (IBA‐1) ( n = 4). I) In situ hybridization RNAscope images of Trpc5 mRNA in neurons (NeuN), astrocytes (GFAP) and microglia (IBA‐1) ( n = 4). J) qPCR revealed decreased mRNA levels of Trpc5 in primary spinal neurons after morphine exposure (200 µM, 14 h) (Unpaired Student's t test, t (10) = 6.031, P = 0.0001, n = 6). K) Immunoblot analysis revealed downregulation of TRPC5 protein levels in primary spinal neurons after morphine exposure (200 µM, 14 h) (Unpaired Student's t test, t (10) = 8.000, P < 0.0001, n = 8). Data are expressed as mean ± SD, * P < 0.05, ** P < 0.01 and *** P < 0.001.

Article Snippet: In the combined application of the RNAscope Multiplex Fluorescent v2 Assay and immunofluorescence, antibodies against IBA‐1 (CST, #17198T; 1:50), NeuN (CST, #24 307; 1:50), and GFAP (Santa Cruz, sc‐6170; 1:50) were utilized.

Techniques: Protein-Protein interactions, Quantitative Proteomics, Saline, Expressing, Sequencing, In Situ Hybridization, RNAscope, Immunohistochemistry, Western Blot, Confocal Microscopy

Journal: Advanced Science

Article Title: Morphine Tolerance Gated through EZH2‐Mediated Suppression of Trpc5 in Spinal GABAergic Interneurons in Male Mice

doi: 10.1002/advs.202507908

Figure Lengend Snippet: Activation of TRPC5 in GABAergic interneurons improves morphine tolerance. A) Intrathecal injection of RLZ (2 µg/10 µL) had no effect on acute morphine analgesia (two‐way ANOVA, drug effect: F (3, 264) = 259.9, P < 0.0001; time effect: F (5, 264) = 75.23, P < 0.0001; drug × time effect: F (15, 264) = 25.5, P < 0.0001, n = 12). B) Intrathecal injection of RLZ (2 µg/10 µL) significantly improved morphine tolerance (two‐way ANOVA, drug effect: F (3, 308) = 579.3, P < 0.0001; time effect: F (6, 308) = 48.87, P < 0.0001; drug × time effect: F (18, 308) = 20.25, P < 0.0001, n = 12). C) Intrathecal injection of BTD (2 µg/10 µL) significantly enhanced acute morphine analgesic effect (two‐way ANOVA, drug effect: F (3, 168) = 583.3, P < 0.0001; time effect: F (5, 168) = 79.89, P < 0.0001, drug × time effect: F (15, 168) = 23.77, P < 0.0001, n = 8). D) Intrathecal injection of BTD (2 µg/10 µL) significantly improved chronic morphine tolerance (two‐way ANOVA, drug effect: F (3, 227) = 409.5, P < 0.0001; time effect: F (6, 227) = 43.39, P < 0.0001, drug × time effect: F (18, 227) = 15.07, P < 0.0001, n = 9). E) Intrathecal injection of AC1903 (2 µg/10 µL) had no effect on acute morphine analgesia (two‐way ANOVA, drug effect: F (3, 264) = 610.2, P < 0.0001; time effect: F (5, 264) = 221.6, P < 0.0001, drug × time effect: F (15, 264) = 78.07, P < 0.0001, n = 12). F) Intrathecal injection of AC1903 (2 µg/10 µL) accelerated chronic morphine tolerance (two‐way ANOVA, drug effect: F (3, 308) = 482.5, P < 0.0001; time effect: F (6, 308) = 157.7, P < 0.0001, drug × time effect: F (18, 308) = 52.80, P < 0.0001, n = 12). G) WT and Trpc5 −/− mice were subjected to morphine (i.t., 10 µg/10 µL) and tail‐flick test was performed 30 min after drugs injection. Trpc5 deficiency weakened the analgesic effects of acute morphine (two‐way ANOVA, drug effect: F (2, 90) = 184.2, P < 0.0001; time effect: F (5, 90) = 98.94, P < 0.0001, drug × time effect: F (10, 90) = 26.11, P < 0.0001, n = 6, Trpc5 −/− + morphine versus WT + morphine). H) WT and Trpc5 −/− mice were subjected to morphine (i.t., 10 µg/10 µL) for 7 consecutive days and tail‐flick test was performed 30 min after drugs injection. Trpc5 deficiency markedly accelerated the development of morphine tolerance (two‐way ANOVA, drug effect: F (2, 105) = 124.6, P < 0.0001; time effect: F (6, 105) = 60.96, P < 0.0001, drug × time effect: F (12, 105) = 19.48, P < 0.0001, Trpc5 −/− + morphine versus WT + morphine). I) Double immunofluorescence showed distinctive TRPC5 (green) expression in GABAergic interneurons (GABA, red) and glutamergic interneurons (VGLUT2, red) in the spinal cord of naïve mice. J) In situ hybridization RNAscope images of Trpc5 mRNA in inhibitory interneurons ( Slc32a1 + ) and excitatory interneurons ( Slc17a6 + ). White arrows indicate Trpc5 double labeled with Slc32a1 ( n = 4). K) Scheme of the pAAV‐mediated Cre recombinase expression (EGFP + , green) in GABAergic interneurons and diphtheria toxin (DT) injection regimen to ablate spinal GABAergic interneurons in R26 LSL‐DTR mice. L) Immunostaining of GAD67 or TRPC5 with EGFP in the spinal dorsal horn of vehicle or DTX‐treated R26 LSL‐DTR mice. White arrows indicate GAD67 or TRPC5 double labeled with EGFP (n = 4). M) Scheme of chronic morphine exposure regimen used for tail‐flick test in mice. Mice were intrathecally injected with pSLenti‐TRPC5 or its control pSLenti‐EGFP before chronic morphine exposure (10 µg/10 µL, 7d). N) Representative photomicrographs with an inset showed pSLenti‐mediated TRPC5 expression (EGFP + , green) in GABAergic interneurons (GABA, red) in the spinal cord of mice. O) TRPC5 overexpression in GABAergic interneurons of spinal cord significantly improved chronic morphine tolerance (two‐way ANOVA, drug effect: F (3, 252) = 726.8, P < 0.0001; time effect: F (6, 252) = 34.27, P < 0.0001, drug × time effect: F (18, 252) = 34.27, P < 0.0001, n = 10). P) ELISA analysis of GABA in CSF from rats exposed to morphine (i.t., 20 µg /10 µL) with or without RLZ (i.t., 4 µg /10 µL) for 7 continuous days (one‐way ANOVA, F (3,38) = 44.16, P < 0.0001, control group: n = 11, morphine group: n = 10, morphine paired with RLZ: n = 11, RLZ group: n = 10). I) Data displayed that RLZ (i.t., 2 µg/10 µL) improved morphine tolerance and Bicuculline (i.t., 5 µg/10 µL) abolished the effect of RLZ on morphine tolerance (two‐way ANOVA, drug effect: F (3, 196) = 450.3, P < 0.0001; time effect: F (6, 196) = 100.2, P < 0.0001, drug × time effect: F (18, 196) = 28.73, P < 0.0001, n = 8). Data are expressed as mean ± SD, * P <0.05, ** P < 0.01 and *** P < 0.001.

Article Snippet: In the combined application of the RNAscope Multiplex Fluorescent v2 Assay and immunofluorescence, antibodies against IBA‐1 (CST, #17198T; 1:50), NeuN (CST, #24 307; 1:50), and GFAP (Santa Cruz, sc‐6170; 1:50) were utilized.

Techniques: Activation Assay, Injection, Tail Flick Test, Immunofluorescence, Expressing, In Situ Hybridization, RNAscope, Labeling, Immunostaining, Control, Over Expression, Enzyme-linked Immunosorbent Assay

Journal: STAR Protocols

Article Title: Protocol for spatial analysis of multiple markers across adjacent tissue sections captured by RNAscope using CellProfiler

doi: 10.1016/j.xpro.2025.103914

Figure Lengend Snippet: Modular pipeline architecture organized by functional category (A) ROI image data input and signal enhancement modules for importing and enhancing RNAscope ROI images; (B) Object identification modules for qualitative and quantitative assessments of nuclear, cytoplasmic, and extracellular localizations with their respective markers; (C) Filtering and classification modules for cell typing and intermarker association quantification; (D) Neighborhood analysis modules for evaluating intermarker associations and marker density; (E) Measurement and visualization modules for spatial and tissue profile assessment. Static modules (gray) required minimal adjustment across tissue types. Variable modules (blue) are slide- and/or tissue-specific, requiring additional customization for specific gene or intermarker associations.

Article Snippet: Scan and visualize RNAscope slides using a pathology imaging system (e.g., Vectra Polaris Automated Quantitative Pathology Imaging System [Akoya Biosciences, Perkin Elmer]), as discussed by Sari et al . 4.

Techniques: Functional Assay, RNAscope, Marker