Journal: Cell reports

Article Title: Targeting the succinate receptor effectively inhibits periodontitis

doi: 10.1016/j.celrep.2022.111389

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Human Gingival Fibroblast cells: HGF-1 , ATCC , CRL-2014.

Techniques: Virus, Recombinant, Blocking Assay, Modification, SYBR Green Assay, RNAscope, Formulation, Reverse Transcription, Enzyme-linked Immunosorbent Assay, Multiplex Assay, Picogreen Assay, Sequencing, Software, Microscopy, Micro-CT

Journal: Neuron

Article Title: Semaphorin 6D tunes amygdalar circuits for emotional, metabolic, and inflammatory outputs.

doi: 10.1016/j.neuron.2024.06.017

Figure Lengend Snippet: Figure 1. Loss of Sema6d increases anxiety (A) Phenome-wide association of SEMA6D polymorphisms across 4,756 GWASs from the GWAS Atlas. (B) SEMA6D mRNA expression in the amygdala from patients with schizophrenia compared with controls in the PRJNA379666 dataset. TPM, transcripts per kilobase million. Boxes denote the interquartile range (IQR). The median is shown as horizontal bars. Whiskers extend to 1.5 times the IQR. Outliers are shown as individual points. Healthy controls, n = 24; patients with schizophrenia, n = 22.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Blocker Casein in PBS ThermoFisher Scientific Cat# 37582 Cholera Toxin Subunit B (Recombinant), Alexa Fluor 594 Conjugate ThermoFisher Scientific Cat# C34777 Corn oil Sigma-Aldrich Cat# C8267 DAPI Solution Thermo fisher Cat# 62248 FD Rapid GolgiStain Kit FD NeuroTechnologies Cat# PK401 Fluoromount Diagnostic BioSystems Cat# K024 Olive oil Wako Cat# 150-00276 OPAL 520 REAGENT PACK Akoya Biosciences Cat# FP1487001KT OPAL 570 REAGENT PACK Akoya Biosciences Cat# FP1488001KT OPAL 690 REAGENT PACK Akoya Biosciences Cat# FP1497001KT RNAscope(R) Multiplex Fluorescent Reagent Kit v2 Advanced Cell Diagnostics, Inc. Cat# 323100 RNAscope(R) Target ProbeMm-Gad2-C3, Mouse Advanced Cell Diagnostics, Inc. Cat# 439371-C3 RNAscope(R) Target ProbeMm-Plxna4-C2, Mouse Advanced Cell Diagnostics, Inc. Cat# 515491-C2 RNAscope(R) Target ProbeMm-Rbfox3-C3, Mouse Advanced Cell Diagnostics, Inc. Cat# 313311-C3 RNAscope(R) Target ProbeMm-Sema6d-C1, Mouse Advanced Cell Diagnostics, Inc. Cat# 565871 RNAscope(R) Target ProbeMm-Sema6d-O1-C1, Mouse Advanced Cell Diagnostics, Inc. Cat# 1255981-C1 Sodium azide Wako Cat# 195-11092 Tamoxifen Sigma-Aldrich Cat# T5648 Triton X-100 Nacalai Tesque Cat# 35501-02 Type I collagenase Gibco Cat# 17100017 RIPA Buffer Nacalai Tesque Cat# 08714-04 4%-Paraformaldehyde Phosphate Buffer Solution Nacalai Tesque Cat# 09154-85 Critical commercial assays Chromium Next GEM Single Cell 3ʹ GEM, Library & Gel Bead Kit v3.1 10x Genomics Cat# 1000128 Chromium Nuclei Isolation Kit with RNase Inhibitor 10x Genomics Cat# 1000494 LIVE/DEAD Fixable Aqua Dead Cell Stain Kit, for 405 nm excitation Invitrogen Cat# L34957 LIVE/DEAD Fixable Violet Dead Cell Stain Kit, for 405 nm excitation Invitrogen Cat# L34955 Myelin removal beads Miltenyi Biotec Cat# 130-096-733 Neural Tissue Dissociation Kit (P) Miltenyi Biotec Cat# 130-092-628 PowerUp SYBR Green Master Mix ThermoFisher Scientific Cat# A25741 QuantiFast Multiplex PCR Kit Qiagen Cat# 204757 RNeasy Plus Mini Kit Qiagen Cat# 74136 SuperScript IV Reverse Transcriptase ThermoFisher Scientific Cat# 18090010 TruSeq Stranded mRNA Library Prep Illumina Cat# 20020594 Visium Spatial for FFPE Gene Expression Kit, Mouse Transcriptome 10x Genomics Cat# 1000339 Deposited data RNA sequencing data This paper GEO: GSE201216 RNA sequencing data This paper GEO: GSE195825 (Continued on next page) e2 Neuron 112, 1–18.e1–e9, September 4, 2024

Techniques: Expressing

Journal: eLife

Article Title: Axon-specific microtubule regulation drives asymmetric regeneration of sensory neuron axons

doi: 10.7554/eLife.104069

Figure Lengend Snippet:

Article Snippet: Tissues were further blocked with 5% normal donkey serum for 30 min. Primary antibodies, namely rabbit anti-KATNA1 (1:500, Proteintech, #17560-1-AP), rabbit anti-CRMP5 (1:200, Abcam, #ab36203), mouse anti-tau (1:100, Cell Signalling, #4019), rabbit anti-βIII-tubulin (1:200, Synaptic Systems, #302302), and mouse anti-βIII-tubulin (1:500, Promega, #G7121) were incubated overnight at 4°C in blocking buffer.

Techniques: Knock-Out, Transfection, Construct, Plasmid Preparation, Transduction, IF-cells, Sequencing, RNAscope, Blocking Assay, Multiplex Assay, Software, Staining, Formulation, Immunofluorescence, Amplification, Live Cell Imaging, Imaging, Western Blot

Journal: eLife

Article Title: Axon-specific microtubule regulation drives asymmetric regeneration of sensory neuron axons

doi: 10.7554/eLife.104069

Figure Lengend Snippet:

Article Snippet: Membranes were either exposed to Fuji Medical X-Ray Film (#16195209, Fujifilm) and scanned using a Molecular Imager GS800 (Bio-Rad) or directly scanned using a ChemiDoc Imaging System (Bio-Rad).

Techniques: Knock-Out, Transfection, Construct, Plasmid Preparation, Transduction, IF-cells, Sequencing, RNAscope, Blocking Assay, Multiplex Assay, Software, Staining, Formulation, Immunofluorescence, Amplification, Live Cell Imaging, Imaging, Western Blot

Journal: ACS Nano

Article Title: Robust, Durable Gene Activation In Vivo via mRNA-Encoded Activators

doi: 10.1021/acsnano.1c10631

Figure Lengend Snippet: In vitro gene activation of B4galnt2 in AML12 cells. (a) Single sgRNA screen by qPCR (top) and flow cytometry (bottom) at 24 h post-transfection. (b) Combinatorial screen of five sgRNAs by qPCR (top) and flow cytometry (bottom) at 24 h post-transfection. (c) Time course of gene activation by qPCR (left) and flow cytometry (right). (d) RNAscope assay against B4galnt2 mRNA (green) and DAPI (blue). Scale bar is 50 μm. B4 denotes B4galnt2 sgRNAs. NT denotes nontargeted sgRNAs. UT denotes untreated mice. RQ (relative quantification) denotes the fold change of B4galnt2 mRNA in treated samples relative to untreated samples and normalized to Gapdh mRNA for both. Data are presented as mean ± SEM ( n = 3 biological replicates). Statistical significance was assessed using a two-way ANOVA followed by Dunnett’s multiple comparison between the nontargeted condition (**** P < 0.0001).

Article Snippet: B4galnt2 gene relative quantification was done by TaqMan primer probe set Mm00484661_m1 (Thermo Fisher Scientific) normalized with the Gapdh gene (Mm99999915_g1) ( Table S5 ).

Techniques: In Vitro, Activation Assay, Flow Cytometry, Transfection, RNAscope, Quantitative Proteomics, Comparison

Journal: ACS Nano

Article Title: Robust, Durable Gene Activation In Vivo via mRNA-Encoded Activators

doi: 10.1021/acsnano.1c10631

Figure Lengend Snippet: In vivo dose optimization of B4galnt2 gene activation. (a) Images of liver sections showing RNAscope staining for B4galnt2 mRNA (green), Dolichos biflorus agglutinin (DBA) lectin staining (magenta), and DAPI (blue) at 1 day postinjection. Scale bar is 25 μm. (b) Flow cytometry plots showing DBA lectin staining of hepatocytes at 48 h using 1 mg/kg VPR mRNA and 1 mg/kg sgRNA. B4galnt2 mRNA copy numbers (c) and the percentage of activated hepatocytes (heps) (d) between formulation approaches at varying mRNA doses with constant sgRNA/mRNA mass ratio. Heat maps of B4galnt2 mRNA copies (e) and the percentage of activated hepatocytes (f) with varying mRNA and sgRNA amounts. (g) Direct measurement of LNP encapsulation of mRNA by qPCR. Data were normalized to the separate LNP formulation condition. Data are presented as mean ± SEM ( n = 4 technical replicates). (h) VPR mRNA copy numbers from liver samples compared to theoretical doses. Linear regression was performed for each data set (solid line = best fit, dotted lines = 95% CI). (i) Overall delivery of VPR mRNA to livers for each formulation approach is reported as the slope of the linear fits from (h). Data were normalized to the separate LNP formulation condition. Data are presented as mean ±95% CI. (j) Dose-corrected qPCR results from combined and separate formulation experiments. (k) Dose-corrected flow cytometry results from combined and separate formulation experiments. Unless otherwise noted, data represent mean ± SEM ( n = 3–4 mice). 4PL curves were fit to data in (d) and (k) (solid lines = best fit curve, dotted lines = 95% CI). An extra sum-of-squares F-test was performed to assess statistical significance between the EC 50 values of 4PL fits and slopes of linear fits. When P > 0.05, a combined EC 50 value was reported for the curves. Additional statistical significance was assessed using a two-way ANOVA followed by Dunnett’s multiple comparison (c,d,h) and a student’s t test (g,i) between formulation approaches (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).

Article Snippet: B4galnt2 gene relative quantification was done by TaqMan primer probe set Mm00484661_m1 (Thermo Fisher Scientific) normalized with the Gapdh gene (Mm99999915_g1) ( Table S5 ).

Techniques: In Vivo, Activation Assay, RNAscope, Staining, Flow Cytometry, Formulation, Encapsulation, Comparison

Journal: ACS Nano

Article Title: Robust, Durable Gene Activation In Vivo via mRNA-Encoded Activators

doi: 10.1021/acsnano.1c10631

Figure Lengend Snippet: In vivo demonstration of optimized B4galnt2 gene activation. (a) Representative slide scan images of liver sections showing RNAscope staining for B4galnt2 mRNA (green) and DAPI (blue). Insets depict the relative locations of 4× and 16× views. Scale bars are 800 μm for 1×, 200 μm for 4×, and 50 μm for 16× images. Time courses of B4galnt2 mRNA (b) and activator mRNA (c) copy numbers from liver tissue over 9 days. B4galnt2 mRNA copy numbers (d) and percentage of activated hepatocytes (e) in mice treated with activator mRNA and B4 sgRNA with or without AcrIIA4 co-delivery. The “–AcrIIA4” groups were dosed with activator mRNA and B4 sgRNA on day 0. The “+AcrIIA4” groups were simultaneously dosed with activator mRNA, B4 sgRNA, and AcrIIA4 mRNA on day 0. All VPR and VPH-SS18-treated mice were euthanized at day 1 postinjection, and all p300-treated mice were euthanized at 5 days postinjection. (f) VPH-SS18 time course with redosing. VPH-SS18 mRNA and B4 sgRNAs were delivered to both groups on day 0. AcrIIA4 treatment was given to one group on day 5, followed by euthanasia on day 6. Remaining mice that did not receive AcrIIA4 mRNA were then redosed with VPH-SS18 mRNA and B4 sgRNAs on day 14. Data represent mean ± SEM ( n = 3–4 mice). Statistical significance was assessed using a two-way ANOVA followed by Dunnett’s multiple comparison compared to the NT-treated group (b,c) and a student’s t test (d,e) (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).

Article Snippet: B4galnt2 gene relative quantification was done by TaqMan primer probe set Mm00484661_m1 (Thermo Fisher Scientific) normalized with the Gapdh gene (Mm99999915_g1) ( Table S5 ).

Techniques: In Vivo, Activation Assay, RNAscope, Staining, Comparison

Journal: Poultry Science

Article Title: Effects of high incubation temperature on tight junction proteins in the yolk sac and small intestine of embryonic broilers

doi: 10.1016/j.psj.2023.102875

Figure Lengend Snippet: In situ hybridization for occludin mRNA in the yolk sac tissue of embryos and day of hatch chicks. Eggs were incubated at 37.5°C (Experiment 2). Yolk sac tissue samples were collected from embryonic d 7 (E7) to day of hatch (DOH). Formalin-fixed paraffin-embedded yolk sac tissue samples ( n = 3) were processed for in situ hybridization using a probe for chicken occludin and the RNAscope 2.5 HD Assay-RED detection kit. Occludin mRNA was revealed as red fluorescence. Sections were counterstained with hematoxylin. Replicate 1 (Rep1) and Rep2 were hybridized at the same time, while Rep3 was done separately. Fluorescent images were captured at 200× with a Nikon Eclipse 80i microscope and a Nikon DS-Ri1 camera. Scale bar equals 100 μm.

Article Snippet: Images were captured using fluorescence with a Nikon Eclipse 80i microscope and Nikon DS-Ri1 or DS-Ri2 cameras.

Techniques: In Situ Hybridization, Incubation, Formalin-fixed Paraffin-Embedded, RNAscope 2.5 HD Assay, Fluorescence, Microscopy

Journal: Poultry Science

Article Title: Effects of high incubation temperature on tight junction proteins in the yolk sac and small intestine of embryonic broilers

doi: 10.1016/j.psj.2023.102875

Figure Lengend Snippet: In situ hybridization for JAMA and JAM2 mRNA in the jejunum and yolk sac tissue of day of hatch chicks. Eggs were incubated at 37.5°C (Experiment 1). Sections of the jejunum and yolk sac tissue from chicks at day of hatch were fixed in formaldehyde and embedded in paraffin. In situ hybridization was performed using probes for chicken JAMA and JAM2 and the RNAscope 2.5 HD Assay-RED. JAMA and JAM2 mRNA were revealed as red fluorescence. Sections were counterstained with hematoxylin. Fluorescent images were captured at 100× with a Nikon Eclipse 80i microscope and a Nikon DS-Ri2 camera. Scale bar equals 100 μm.

Article Snippet: Images were captured using fluorescence with a Nikon Eclipse 80i microscope and Nikon DS-Ri1 or DS-Ri2 cameras.

Techniques: In Situ Hybridization, Incubation, RNAscope 2.5 HD Assay, Fluorescence, Microscopy

Journal: Cell

Article Title: Microbial exposure during early human development primes fetal immune cells

doi: 10.1016/j.cell.2021.04.039

Figure Lengend Snippet:

Article Snippet: Human IL-2 IS premium grade , Miltenyi Biotec , Cat# 130-097-745.

Techniques: Flow Cytometry, Formalin-fixed Paraffin-Embedded, Staining, Modification, Membrane, RNAscope, Multiplex Assay, Sequencing, Gene Expression, Software, Imaging