Journal: Cell Reports Methods

Article Title: A genetic tool for the longitudinal study of a subset of post-inflammatory reactive astrocytes

doi: 10.1016/j.crmeth.2022.100276

Figure Lengend Snippet: Expression of Lcn2CreERT2 following repeated low-dose LPS treatment (A) Immunostaining for tdTomato in Lcn2CreERT2 ; Ai9 brains. Animals were given one of the following four treatments for 5 consecutive days: Saline and corn oil, saline and tamoxifen, LPS and corn oil, LPS and tamoxifen. Sections were counterstained with DAPI. (B) Quantification of total number of red cells/section from animals in (A). ∗∗∗p < 0.001 ANOVA and Tukey’s post hoc test, n = 5 animals per treatment. (C) Immunostaining of brains from saline- (left) or LPS-treated (right) Lcn2CreERT2 ; Ai9 animals for tdTomato and the astrocytic marker GFAP (top), the endothelial and microglial marker lectin (second row), the neuronal marker NeuN (third row), and the microglial marker Iba1 (bottom). Sections were counterstained with DAPI. White arrowheads indicate co-positive cells for tdTomato and the panel maker. (D) Quantification of co-positive cells from (C). (E) RNAscope for the astrocytic marker Slc1a3 (top) and the endothelial marker Pecam1 (bottom) on brains from LPS- and saline-treated Lcn2CreERT2 ; Ai9 animals. Sections were immunostained for tdTomato and counterstained with DAPI. (F) Quantification of cells co-positive for tdTomato and RNA markers described in (E). Scale bars, (A) 250 μm, (C) 50 μm, (E) 25 μm. Error bars ± SEM. See also Figures S1 and .

Article Snippet: RNAScope Multiplex Fluorescent Detection kit v2 , Advanced Cell Diagnostics , 323110.

Techniques: Expressing, Immunostaining, Saline, Marker, RNAscope

Journal: Cell Reports Methods

Article Title: A genetic tool for the longitudinal study of a subset of post-inflammatory reactive astrocytes

doi: 10.1016/j.crmeth.2022.100276

Figure Lengend Snippet: Astrocytes that were initially reactive remain in the brain after resolution of inflammation (A) Lcn2CreERT2 ; Ai9 brain sections immunostained for tdTomato and counterstained with DAPI. Animals were given tamoxifen and saline or LPS and collected at either 24 h or 1 month after the last treatment. (B) Quantification of tdTomato-positive cells in the thalamic region. p = 0.38, ANOVA followed by Tukey’s HSD post hoc, n = 5 animals. (C) RNAScope on Lcn2CreERT2 ; Ai9 brain sections for the astrocyte marker Aldh1l1 24 h and 1 month after low-dose LPS treatment, followed by immunostaining for tdTomato and counterstaining with DAPI. (D) Quantification of Aldh1l1 and tdTomato co-positive cells in (C). p = 0.16, paired t test. n = 18 pooled sections across three animals. (E) Co-labeling of tdTomato and EdU-positive astrocytes in Lcn2CreERT2 ; Ai9 animals after LPS treatment. (F) High magnification image of labeled astrocytes and EdU-positive cells at 1 month. (G) Quantification of tdTomato and EdU co-positive astrocytes at 24 h, 7 days, and 1 month following LPS treatment. Error bars ± SEM. Scale bars, (A) 400 μm, (C, E) 200 μm, (F) 20 μm. See also Figure S4 .

Article Snippet: RNAScope Multiplex Fluorescent Detection kit v2 , Advanced Cell Diagnostics , 323110.

Techniques: Saline, RNAscope, Marker, Immunostaining, Labeling

Journal: Cell Reports Methods

Article Title: A genetic tool for the longitudinal study of a subset of post-inflammatory reactive astrocytes

doi: 10.1016/j.crmeth.2022.100276

Figure Lengend Snippet: Reactive marker expression decreases 1 month after inflammation (A and B) Analysis of reactive marker expression within tdTomato-positive cells. RNAScope for brain sections 24 h after LPS, or 1 month after LPS treatment to detect expression of the reactive markers Lcn2 (A) and GFAP (B), followed by immunostaining for tdTomato and counterstaining with DAPI. RNAScope for reactive markers Lcn2 (A) and GFAP (B) was quantified by looking at both number of fluorescent puncta (A, B, top panels) and integrated fluorescent density (A, B, bottom panels). ∗∗∗p < 0.001, paired t test. n = 29 astrocytes pooled from three animals. Error bars ± SEM. Scale bars, 50 μm. See also Figure S5 .

Article Snippet: RNAScope Multiplex Fluorescent Detection kit v2 , Advanced Cell Diagnostics , 323110.

Techniques: Marker, Expressing, RNAscope, Immunostaining

Journal: Cell Reports Methods

Article Title: A genetic tool for the longitudinal study of a subset of post-inflammatory reactive astrocytes

doi: 10.1016/j.crmeth.2022.100276

Figure Lengend Snippet: Reactive markers return to baseline levels in astrocytes that were initially reactive (A–C) RNAScope staining (green) for Slc1a2 (A), Slc1a3 (B), and Lrrc8a (C) in Lcn2CreERT2 ; Ai9 animals treated with saline or LPS. Sections were immunostained for tdTomato and counterstained with DAPI. Bottom: quantification of RNAScope puncta per tdTomato + astrocyte. ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, ANOVA followed by Tukey’s honest significant difference post hoc test. n = 30 pooled astrocytes across three animals. Error bars ± SEM. Scale bars, 20 μm.

Article Snippet: RNAScope Multiplex Fluorescent Detection kit v2 , Advanced Cell Diagnostics , 323110.

Techniques: RNAscope, Staining, Saline

Journal: Cell Reports Methods

Article Title: A genetic tool for the longitudinal study of a subset of post-inflammatory reactive astrocytes

doi: 10.1016/j.crmeth.2022.100276

Figure Lengend Snippet:

Article Snippet: RNAScope Multiplex Fluorescent Detection kit v2 , Advanced Cell Diagnostics , 323110.

Techniques: Virus, Recombinant, Plasmid Preparation, RNAscope, Multiplex Assay

Journal: JCI Insight

Article Title: The XCL1/XCR1 axis is upregulated in type 1 diabetes and aggravates its pathogenesis

doi: 10.1172/jci.insight.178743

Figure Lengend Snippet: ( A ) Volcano blots of the gene expression profile of chemokine ligands and their receptors of laser-dissected islets from RIP-GP mice at day 1, 3, 7, 10, 14, and 28 after the LCMV infection in comparison with uninfected mice. XCL1 is highlighted in red and XCR1 in blue. ( B ) Quantification of the data obtained by gene array (curves) and reverse transcription PCR (RT-PCR) (bars) of laser-dissected islets for the expression of XCL1 and XCR1. Data are shown as mean ± SD. ( C ) Duplex RNAScope in situ hybridization XCL1 (red) and XCR1 (blue) of pancreas tissue sections from RIP-GP mice at days 0, 7, 10, 14, 28 after infection ( n = 3 mice per time point). Representative images are displayed per time. Original magnification, 63× (oil). Scale bar: 20 μm. ( D ) Duplex RNAScope in situ hybridization for XCL1 (red) and XCR1 (blue) of pancreas tissue sections from NOD mice obtained at different age and disease state ( n = 2–3). Mice were considered diabetic with nonfasting BG levels of > 300 mg/dL. Original magnification, 40×. Scale bars: 25 μm. ( E ) Quantification of XCL1 (left) and XCR1 (right) expression in the islets of RIP-GP mice at day 0, 7, 10, 14, and 28 after infection ( n = 3 per time point). Data are shown as mean ± SEM, and significant P values (2-way ANOVA) are indicated. ( F ) Quantification of XCL1 (left) and XCR1 (right) expression in the islets of NOD mice at different time points ( n = 2–3 per time point). Data are shown as mean ± SEM.

Article Snippet: The human pancreas sections have been tested for RNA integrity sufficient for the RNAScope duplex detection method (ACD/Bio-Techne) using positive control probe sets provided by the supplier.

Techniques: Gene Expression, Infection, Comparison, Reverse Transcription, Reverse Transcription Polymerase Chain Reaction, Expressing, RNAscope, In Situ Hybridization

Journal: JCI Insight

Article Title: The XCL1/XCR1 axis is upregulated in type 1 diabetes and aggravates its pathogenesis

doi: 10.1172/jci.insight.178743

Figure Lengend Snippet: Human pancreas slides were obtained through the HPAP program. Groups of individuals were divided as follows: nondiabetic (ND), nondiabetic with known T1D familiarity (T1D familiarity), autoantibody positive (Aab + ), and patients with diabetes (T1D). ( A ) Representative pictures of duplex RNAScope in situ hybridization for XCL1 (red) and XCR1 (blue). In the left column, the original picture with a magnified example of a positive cell (square). On the right, XCR1-expressing cells are highlighted with a light blue dot, and XCL1-expressing cells are highlighted with a red dot. Islets are indicated with black lines. Original magnification, 63×. Scale bars: 20 μm. ( B ) Number of XCR1-expressing cells per islet microenvironment. Number of islets per section analyzed was 6–63. Each islet is represented by 1 dot. Data are shown as the mean ± SEM number of XCR1-expressing cell in one individual. ( C ) Mean ± SEM of XCR1-expressing cells per islet microenvironment in the groups of ND, T1D familiarity, Aab + , and T1D. Number of islets per section analyzed was 6–63. The mean for every individual is represented by dots.

Article Snippet: The human pancreas sections have been tested for RNA integrity sufficient for the RNAScope duplex detection method (ACD/Bio-Techne) using positive control probe sets provided by the supplier.

Techniques: RNAscope, In Situ Hybridization, Expressing

Journal: JCI Insight

Article Title: The XCL1/XCR1 axis is upregulated in type 1 diabetes and aggravates its pathogenesis

doi: 10.1172/jci.insight.178743

Figure Lengend Snippet: ( A ) Duplex RNAScope in situ hybridization for XCL1 (red) and XCR1 (blue) of pancreas tissue sections obtained from RIP-GP × XCL1 –/– mice at different times after infection, demonstrating that XCL1-deficient mice do not express XCL1. Original magnification, 63× (oil). Scale bars: 20 μm. ( B ) IHC staining for CD103 of quick-frozen pancreas sections at different times after LCMV infection, comparing RIP-GP with RIP-GP × XCL1 –/– mice. Representative pictures are shown. Original magnification, 40×. Scale bars: 25 μm. ( C ) Immunofluorescence double-staining for CD11c (green) and CD103 (red) of quick-frozen pancreas sections obtained at different times after LCMV infection, comparing RIP-GP with RIP-GP × XCL1 –/– mice. Nuclei are stained with DAPI (blue). White arrows indicate the double-positive cells (cDC1). Representative pictures are shown. Original magnification, 40×. Scale bars: 20 μm. ( D ) Quantification of the CD103 staining in RIP-GP (light gray) and RIP-GP × XCL1 –/– (dark gray) mice shown in B , expressed as a percentage of positive CD103 cell area per islet area. Numbers of mice per group are indicated. Data are shown as mean ± SEM. ( E ) Quantification of the CD103/CD11c double-staining in RIP-GP (light gray) and RIP-GP × XCL1 –/– (dark gray) mice shown in C , expressed as a percentage of CD103/CD11c–double-positive cell area per CD11c + cell area. Numbers of mice per group are indicated. Data are shown as mean ± SEM, and significant P values (Mann-Whitney t test) are indicated.

Article Snippet: The human pancreas sections have been tested for RNA integrity sufficient for the RNAScope duplex detection method (ACD/Bio-Techne) using positive control probe sets provided by the supplier.

Techniques: RNAscope, In Situ Hybridization, Infection, Immunohistochemistry, Immunofluorescence, Double Staining, Staining, MANN-WHITNEY

Journal: American Journal of Physiology - Renal Physiology

Article Title: Unique miRNome and transcriptome profiles underlie microvascular heterogeneity in mouse kidney

doi: 10.1152/ajprenal.00005.2023

Figure Lengend Snippet: miRNAscope target probes for detection of miRNA via in situ hybridization in mouse samples

Article Snippet: ISH was performed using miRNAscope HD Reagent kit RED (Advanced Cell Diagnostics, Newark, CA) on 10-μm-thick fresh-frozen mouse kidney sections according to the manufacturer’s instructions, with the exception of tissue fixation in 4% (wt/vol) paraformaldehyde (Sigma-Aldrich)-PBS (Thermo Fisher Scientific), which was extended to 2 h instead of the recommended 1 h. Selected miRNAs were detected using target-specific miRNAscope probes (Advanced Cell Diagnostics) ( ).

Techniques: In Situ Hybridization, Positive Control, Negative Control

Journal: iScience

Article Title: Accessing genetically defined cell types in the superior colliculus with transgenic mouse lines

doi: 10.1016/j.isci.2025.112194

Figure Lengend Snippet:

Article Snippet: RNAscope HiPlex12 Reagent Kit v2 (488, 550, 650) , Advanced Cell Diagnostics , Cat #: 324419.

Techniques: Recombinant, Single Cell, RNAscope, Multiplex Assay, RNA Sequencing, Mutagenesis, Software