Journal: Frontiers in Immunology

Article Title: Development and characterization of segment-specific enteroids from the pig small intestine in Matrigel and transwell inserts: insights into susceptibility to porcine epidemic diarrhea Virus

doi: 10.3389/fimmu.2024.1451154

Figure Lengend Snippet: Summary of the antibodies used in the immunohistochemistry and immunocytochemistry in porcine tissues, porcine enteroids Matrigel culture (PEMCs) and porcine enteroids transwell culture (PETCs) in this study.

Article Snippet: Novus Biologicals , LGR5 , NBP1-28904SS , – , – , Rabbit polyclonal , 2 µg/ml.

Techniques: Immunohistochemistry, Immunocytochemistry, Concentration Assay

Journal: Frontiers in Immunology

Article Title: Development and characterization of segment-specific enteroids from the pig small intestine in Matrigel and transwell inserts: insights into susceptibility to porcine epidemic diarrhea Virus

doi: 10.3389/fimmu.2024.1451154

Figure Lengend Snippet: Summary of various cell marker expressions (immunohistochemistry/immunocytochemistry and gene expression) in the tissues, PEMCs and PETCs derived from 3 regions (duodenum, jejunum, and ileum) of the porcine small intestine.

Article Snippet: Novus Biologicals , LGR5 , NBP1-28904SS , – , – , Rabbit polyclonal , 2 µg/ml.

Techniques: Marker, Immunohistochemistry, Expressing, Derivative Assay

Journal: Frontiers in Immunology

Article Title: Development and characterization of segment-specific enteroids from the pig small intestine in Matrigel and transwell inserts: insights into susceptibility to porcine epidemic diarrhea Virus

doi: 10.3389/fimmu.2024.1451154

Figure Lengend Snippet: Cellular characterization of paraffin-embedded day 5-7 porcine enteroids in Matrigel culture (PEMCs) cross segments derived from three porcine small intestine regions (duodenum, jejunum, ileum) at 400X magnification (cropped images). (A1–A3) Epithelial cell marker pan-cytokeratin. (B1–B3) Brush border of the enterocyte marker villin. (C1–C3) Periodic acid-Schiff (PAS) stain showing magenta red for neutral mucins. (D1–D3) Alcian blue stain demonstrating acidic mucins. (E1–E3) Neuroendocrine cell marker Chromogranin A. (F1–F3) Paneth cell marker lysozyme C. (G1–G3) Cell proliferation marker PCNA. (H1–H3) Stem cell marker LGR5. (I1 to I3) Tight-junction marker ZO-1. (J1–J3) Adheren-junction marker E-Cadherin. (K1–K3) Tight-junction marker Occludin.

Article Snippet: Novus Biologicals , LGR5 , NBP1-28904SS , – , – , Rabbit polyclonal , 2 µg/ml.

Techniques: Derivative Assay, Marker, Staining

Journal: Frontiers in Immunology

Article Title: Development and characterization of segment-specific enteroids from the pig small intestine in Matrigel and transwell inserts: insights into susceptibility to porcine epidemic diarrhea Virus

doi: 10.3389/fimmu.2024.1451154

Figure Lengend Snippet: Cellular characterization of paraffin-embedded day 5-7 porcine enteroids on transwell culture (PETCs) cross segments derived from three porcine small intestine regions (duodenum, jejunum, ileum) at 400X magnification (cropped images). (A1–A3) Epithelial cell marker pan-cytokeratin. (B1–B3) Brush border of the enterocyte marker villin. (C1–C3) Periodic acid-Schiff (PAS) stain showing magenta red for neutral mucins. (D1–D3) Alcian blue stain demonstrating acidic mucins. (E1–E3) Neuroendocrine cell marker Chromogranin A. (F1–F3) Paneth cell marker lysozyme C. (G1–G3) Cell proliferation marker PCNA. (H1–H3) Stem cell marker LGR5. (I1–I3) Tight-junction marker ZO-1. (J1–J3) Adheren-junction marker E-Cadherin. (K1–K3) Tight-junction marker Occludin.

Article Snippet: Novus Biologicals , LGR5 , NBP1-28904SS , – , – , Rabbit polyclonal , 2 µg/ml.

Techniques: Derivative Assay, Marker, Staining

Journal: Frontiers in Immunology

Article Title: Development and characterization of segment-specific enteroids from the pig small intestine in Matrigel and transwell inserts: insights into susceptibility to porcine epidemic diarrhea Virus

doi: 10.3389/fimmu.2024.1451154

Figure Lengend Snippet: Cellular characterization of paraffin-embedded tissue cross segments derived from three porcine small intestine regions (duodenum, jejunum, ileum) of 7-10 day-old cesarean-derived, colostrum-deprived (CD/CD) neonatal piglets at 400X magnification (cropped images). (A1–A3) Epithelial cell marker pan-cytokeratin. (B1–B3) Brush border of the enterocyte marker villin. (C1–C3) Periodic acid-Schiff (PAS) stain showing magenta red for neutral mucins. (D1–D3) Alcian blue stain demonstrating acidic mucins. (E1–E3) Neuroendocrine cell marker Chromogranin A. (F1–F3) Paneth cell marker lysozyme C. (G1–G3) Cell proliferation marker PCNA. (H1–H3) Stem cell marker LGR5. (I1–I3) Tight-junction marker ZO-1. (J1–J3) Adheren-junction marker E-Cadherin. (K1–K3) Tight-junction marker Occludin.

Article Snippet: Novus Biologicals , LGR5 , NBP1-28904SS , – , – , Rabbit polyclonal , 2 µg/ml.

Techniques: Derivative Assay, Marker, Staining

Journal: eNeuro

Article Title: Slow NMDA-Mediated Excitation Accelerates Offset-Response Latencies Generated via a Post-Inhibitory Rebound Mechanism

doi: 10.1523/ENEURO.0106-19.2019

Figure Lengend Snippet: Primary and secondary antibodies used for immunocytochemistry

Article Snippet: VGLUT3 , Peptide (C)ELNHEAFVSPRKK, corresponding to amino acid residues 533–545 of rat VGLUT3 (accession Q7TSF2); cytoplasmic, C terminus , Alomone Labs , AGC-037 , Rabbit , 1:300.

Techniques: Clone Assay, Recombinant, Purification, Isolation, Strep-tag

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Single-molecule imaging of the functional crosstalk between surface NMDA and dopamine D1 receptors

doi: 10.1073/pnas.1310145110

Figure Lengend Snippet: The D1R–NMDAR interaction bidirectionally regulates the surface distribution and dynamics of D1R and NMDAR. (A) Immunostaining of surface D1R-CFP (green) and GluN1 subunit (red) in hippocampal neurons. The yellow arrow shows overlay. (B) Immunostaining of surface D1R-CFP in control or after D1/5R agonist, TAT-t2, or TAT-[N2A15] application. (Scale bar, 250 nm.) (C) Normalized measures of D1R-CFP clusters intensity in control (n = 32 neuronal fields), D1/5R agonist-treated (n = 24 neuronal fields; *P < 0.05 compared with control), TAT-NSt2–treated (non-sense of TAT-t2, n = 19 neuronal fields), TAT-t2–treated (n = 21 neuronal fields; **P < 0.01 compared with TAT-NSt2), TAT-NSt3–treated (non-sense of TAT-t3, n = 11 neuronal fields), TAT-t3–treated (n = 12 neuronal fields; P > 0.05 compared with TAT-NSt3), TAT-[NS15]–treated (n = 27 neuronal fields; P > 0.05), or TAT-[N2A15]–treated (n = 21 neuronal fields, *P < 0.05 compared with TAT-[NS15]) conditions. (D) Representative trajectories (1,000 frames, 20-Hz acquisition rate) of surface single D1R-CFP (Left) (green) (scale bar, 400 nm) and GluN1-NMDAR (Right) (blue) (scale bar, 300 nm) in the absence and presence of either D1/5R agonist (10 µM, 15 min) or TAT-t2 (10 µM, 15 min). Bold dotted line, perisynaptic area; thin dotted line, PSD area. (E) Plot of the MSD of surface D1R-CFP (Upper) (green) and GluN1-NMDAR (Lower) (blue) versus time in presence of TAT-NS or TAT-t2 peptides (10 µM, 15 min). The SEM is included for each data point (D1R: TAT-NS, n = 986 trajectories, and TAT-t2, n = 1,326; GluN1-NMDAR: TAT-NS, n = 198, and TAT-t2, n = 134). (F and G) Representative surface distributions of single D1R-CFP (green) (F) and GluN1-NMDAR (blue) (G) in the synaptic area (PSD + perisynaptic area) in control, D1/5R agonist, and TAT-t2 conditions. Each dot represents the detection of a single receptor during a frame. Comparisons of the time spent in the synaptic area (dwell time) by single D1R-CFP (control, n = 173 trajectories; D1/5R agonist, n = 142, **P < 0.01; TAT-t2, n = 752, *P < 0.05) (F) and GluN1-NMDAR (control, n = 189 trajectories; D1/5R agonist, n = 157, *P < 0.05; TAT-t2, n = 134, **P < 0.01) (G) and the synaptic fraction of detected single D1R-CFP (control, n = 14 neuronal fields; D1/5R agonist, n = 19, **P < 0.01; D1/5R agonist in the presence of dynasore, n = 47, **P < 0.01; TAT-t2, n = 15, ***P < 0.001) (F), D5R-CFP (n = 16, P > 0.05) (F), and GluN1-NMDAR (control, n = 11; D1/5R agonist, n = 15, *P < 0.05; TAT-t2, n = 14, *P < 0.05) (G). Dyn., dynasore; D1/5 ago., D1/5 receptor agonist SKF-38393.

Article Snippet: For single-nanoparticle tracking, QD 655 coupled to goat anti-rabbit F(ab′) 2 or anti-mouse IgG (Invitrogen) was incubated (1:10,000, 10 min) onto neurons previously exposed for 10 min to either mouse monoclonal anti-GFP (1 µg; Invitrogen), rabbit polyclonal anti-D1R (1 µg; Lifespan Biosciences), mouse monoclonal anti-GluA2:00 AMPAR subunit (1 µg; Millipore), or rabbit polyclonal anti–GluN1-NMDAR subunit (1 µg; Alomone Laboratories) antibodies.

Techniques: Immunostaining

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Single-molecule imaging of the functional crosstalk between surface NMDA and dopamine D1 receptors

doi: 10.1073/pnas.1310145110

Figure Lengend Snippet: D1R activation or D1R/GluN1-NMDAR interaction blockade increases synaptic NMDAR content and favors AMPAR synaptic long-term potentiation. (A) (Left) Excitatory postsynaptic current traces recorded at −70 mV and +40 mV from a representative hippocampal CA1 pyramidal cell, before and 10 min after exposure to D1/5R agonist. (Right) Relative change over time of the AMPA/NMDA ratio at CA1 synapses in the absence or presence of D1/5R agonist (n = 13, *P < 0.05 10 min after agonist) and in the absence or presence of vehicle (n = 7, P > 0.05). (B) Surface imaging of GluN1-SEP in neurons incubated with either TAT-NS or TAT-t2 (10 µM). (Scale bar, 5 µm.) (Right) Average value of GluN1-SEP content in the synaptic area after TAT-NS or TAT-t2 application (n = 8 neurons per group, **P < 0.01). (C) Dendritic fragment of a hippocampal neuron expressing Homer 1c-DsRed (Upper) and GluA1-SEP (Lower). SEP only fluoresces at neutral pH when receptors are inserted at the plasma membrane. Ten minutes after chemical LTP induction (cLTP), the GluA1-SEP fluorescence intensity increased in postsynaptic clusters. (Insets) High magnification of a synaptic GluA1-SEP cluster. (Scale bar, 2 µm.) (D) Comparison of the synaptic GluA1-SEP fluorescence intensity before and after cLTP with prior TAT-NS (n = 198 synapses, *P < 0.05) or TAT-t2 (n = 215 synapses, *P < 0.05) (TAT-NS versus TAT-t2; *P < 0.05) application. (E) Schematic model of the D1R–NMDAR surface interplay in hippocampal neurons. D1Rs are highly diffusive at the neuronal surface and are dynamically retained in clusters in the vicinity of glutamate synapses where they interact with NMDAR. Dopamine release disrupts this interaction and favors the lateral redistribution of both receptors: D1Rs freely explore extrasynaptic areas, whereas NMDARs laterally reach the PSD where they impact on the long-term plasticity of glutamate synapses.

Article Snippet: For single-nanoparticle tracking, QD 655 coupled to goat anti-rabbit F(ab′) 2 or anti-mouse IgG (Invitrogen) was incubated (1:10,000, 10 min) onto neurons previously exposed for 10 min to either mouse monoclonal anti-GFP (1 µg; Invitrogen), rabbit polyclonal anti-D1R (1 µg; Lifespan Biosciences), mouse monoclonal anti-GluA2:00 AMPAR subunit (1 µg; Millipore), or rabbit polyclonal anti–GluN1-NMDAR subunit (1 µg; Alomone Laboratories) antibodies.

Techniques: Activation Assay, Imaging, Incubation, Expressing, Fluorescence

Journal: JCI Insight

Article Title: TRPC3-Nox2 complex mediates doxorubicin-induced myocardial atrophy

doi: 10.1172/jci.insight.93358

Figure Lengend Snippet: (A and B) Decreases in heart weight (A) and cross-sectional area (CSA) of cardiomyocytes (B) in C57BL/6J mouse hearts 2 weeks after administration of vehicle (Sham) or DOX (n = 6). Scale bar: 50 μm. (C) Abundances of TRPC3 proteins in DOX-treated C57BL/6J mouse hearts (left) and 129Sv-background TRPC3+/+ and TRPC3–/– mouse hearts (right) (n = 3). (D–G) Heart weights (D), CSA (E), and LV contractility (F and G) in 129Sv-background TRPC3+/+ and TRPC3–/– mice treated with vehicle (Sham) or DOX (n = 6). (H) MDA concentrations in mouse hearts (n = 4). (A–H) Data are shown as the mean ± SEM. Significance was determined using unpaired t test analysis in A–C and one-way ANOVA followed by Tukey’s comparison test in D–H. *P < 0.05, **P < 0.01.

Article Snippet: TRPC3 was detected with rabbit anti-TRPC3 antibody (1:1,000, ACC-016, Alomone).

Techniques:

Journal: JCI Insight

Article Title: TRPC3-Nox2 complex mediates doxorubicin-induced myocardial atrophy

doi: 10.1172/jci.insight.93358

Figure Lengend Snippet: (A) Representative fluorescence images (left) and semiquantitative results of fluorescence intensities (right) in mouse hearts stained with hypoxyprobe (n = 3). Scale bar: 40 μm. (B and C) Western blots (B) and quantitative results (C) for Nox2, HIF1α, HO-1, and GAPDH in C57BL/6J mouse hearts 2 weeks after DOX (n = 3). (D) Relationship between heart weight and Nox2 abundance. (E and F) Representative Western blots (E) and quantitative results (F) for Nox2, HIF1α, and GAPDH in 129Sv-background TRPC3+/+ and TRPC3–/– mouse hearts (n = 3). (G) Relationship between heart weight and Nox2 abundance. Data are shown as the mean ± SEM. Significance was determined using unpaired t test analysis in A and C, and one-way ANOVA followed by Tukey’s comparison test in F. *P < 0.05.

Article Snippet: TRPC3 was detected with rabbit anti-TRPC3 antibody (1:1,000, ACC-016, Alomone).

Techniques: Fluorescence, Staining, Western Blot

Journal: JCI Insight

Article Title: TRPC3-Nox2 complex mediates doxorubicin-induced myocardial atrophy

doi: 10.1172/jci.insight.93358

Figure Lengend Snippet: (A and B) Effects of TRPC3-knockdown on DOX-induced cell shrinkage determined by phalloidin staining (A), and ROS production (B) in NRCMs. NRCMs were treated with DOX (3 μM) for 12 h. Cell areas were analyzed using phalloidin staining (n = 3). Scale bars: 20 μm (A); 40 μm (B). (C) Schema for competitive disruption of the TRPC3-Nox2 protein complex by TRPC3 C-terminal minipeptide (C3-C-GFP or C3-C-HA). (D–F) Effects of C3-C-GFP on DOX-induced cardiomyocyte atrophy (D), C3-C-HA on ROS production (E), and C3-C-GFP on Nox2 upregulation and interaction between TRPC3 and Nox2 (F) in H9c2 rat cardiac myoblasts (n = 3-4). Data are shown as the mean ± SEM. Significance was determined using one-way ANOVA followed by Tukey’s comparison test. *P < 0.05.

Article Snippet: TRPC3 was detected with rabbit anti-TRPC3 antibody (1:1,000, ACC-016, Alomone).

Techniques: Staining

Journal: JCI Insight

Article Title: TRPC3-Nox2 complex mediates doxorubicin-induced myocardial atrophy

doi: 10.1172/jci.insight.93358

Figure Lengend Snippet: (A–E) Effects of C3-C-GFP on DOX-induced reduction of heart weight (A), LV systolic (B and C) and diastolic (D) functions, and MDA production (E) in C57BL/6J mouse hearts (n = 3–6). (F) Representative fluorescence images of mouse hearts with or without adenoassociated virus (AAV) encoding C3-C-GFP. Scale bar: 1 mm. (G) Effects of C3-C-GFP on protein abundances of Nox2 and TRPC3 in DOX-treated mouse hearts (n = 3). Data are shown as the mean ± SEM. Significance was determined using one-way ANOVA followed by Tukey’s comparison test. *P < 0.05, **P < 0.01.

Article Snippet: TRPC3 was detected with rabbit anti-TRPC3 antibody (1:1,000, ACC-016, Alomone).

Techniques: Fluorescence

Journal: JCI Insight

Article Title: TRPC3-Nox2 complex mediates doxorubicin-induced myocardial atrophy

doi: 10.1172/jci.insight.93358

Figure Lengend Snippet: (A) Effects of TRPC3 inhibitors on DOX-induced Nox2 upregulation in NRCMs. NRCMs were treated with the indicated TRPC3 inhibitor (1 μM) 30 min prior to DOX treatment (3 μM for 12 h, n = 5). (B–D) Effects of pyrazole-3 (Pyr3) on DOX-induced atrophy (B) and ROS production (C), and upregulation of Nox2 and HIF1α proteins (D) in NRCMs. NRCMs were treated with Pyr3 (1 μM) 30 min prior to DOX (3 μM for 12 h, n = 3). Scale bar: 20 μm (B); 40 μm (C). (E) Abundances of Nox2 proteins in NRCMs with or without Pyr3 treatment for 36 h (n = 3). (F) Effect of Pyr3 (10 μM) on endogenous interaction between TRPC3 and Nox2 in NRCMs (n = 3). (G) Expression levels of TRPC3 and Nox2 mRNAs in NRCMs with or without Pyr3 treatment for 36 h (n = 4). (H and I) Effect of Pyr3 on the interaction between Nox2 and either TRPC3-EGFP or C3-C-GFP in cell-free system. Representative Western blot (H) and quantification of TRPC3-EGFP interacting with Nox2 (I) (n = 3). (J–L) Effect of Pyr3 on DOX-induced LV dysfunction in C57BL/6J mice (n = 6–7). (M) Effect of Pyr3 on DOX-induced MDA production (n = 3). Data are shown as the mean ± SEM. Significance was determined using one-way ANOVA followed by Tukey’s comparison test. *P < 0.05, **P < 0.01.

Article Snippet: TRPC3 was detected with rabbit anti-TRPC3 antibody (1:1,000, ACC-016, Alomone).

Techniques: Expressing, Western Blot

Journal: JCI Insight

Article Title: TRPC3-Nox2 complex mediates doxorubicin-induced myocardial atrophy

doi: 10.1172/jci.insight.93358

Figure Lengend Snippet: (A) Levels of TRPC3 mRNA expression in DOX-treated C57BL/6J mouse hearts (n = 4) and hypoxia-exposed NRCMs (n = 6). (B) Abundances of HO-1 and tubulin in NRCMs exposed to 10% or 1% hypoxia with or without Pyr3 (300 nM, n = 3). (C) Effects of Pyr3 on hypoxia-induced Nox2 upregulation (n = 3). (D) Effects of siRNAs for TRPC3 or Nox2 on hypoxia-induced ROS production. NRCMs were exposed to 1% hypoxia for 12 h and then treated with dihydroethidium (DHE, 2 μM) for 1 h. NRCMs were transfected with siRNAs 72 h before hypoxia (n = 3). Scale bar: 50 μm. Data are shown as the mean ± SEM. Significance was determined using one-way ANOVA followed by Tukey’s comparison test. *P < 0.05, **P < 0.01.

Article Snippet: TRPC3 was detected with rabbit anti-TRPC3 antibody (1:1,000, ACC-016, Alomone).

Techniques: Expressing, Transfection

Journal: JCI Insight

Article Title: TRPC3-Nox2 complex mediates doxorubicin-induced myocardial atrophy

doi: 10.1172/jci.insight.93358

Figure Lengend Snippet: (A) Representative LV pressure-volume loops after inferior vena cava occlusion in TRPC3+/+ and TRPC3–/– mice. (B and C) Results of LV systolic indexes (end-systolic elastance; Ees) (B) and diastolic indexes (end-diastolic pressure-volume relationship β; EDPVRβ) (C) (n = 5). (D) Cardiac performance under volume overload reflecting the Frank-Starling law in TRPC3+/+ and TRPC3–/– mice (n = 6). (E) Cardiac performance under volume overload in C57BL/6J mice treated with Pyr3 (n = 4). (F) Spontaneous walking activity in C57BL/6J mice with and without a running wheel (n = 4). (G–J) Effects of voluntary exercise on heart weight (G) (n = 10), LV performance under volume load (H) (n = 7), and protein abundances of TRPC3, Nox2, HIF1α, and HO-1 (I) (n = 3–4), and relationship between heart weight and Nox2 expression (J). Data are shown as the mean ± SEM. Significance was determined using unpaired t tests in B, C, F, and G and two-way ANOVA followed by Bonferroni’s comparison test in D, E, and H. *P < 0.05, **P < 0.01. (K) Schema for the role of TRPC3 channels in cardiac plasticity. The TRPC3-Nox2 axis restricts volume-loaded LV diastolic filling through ROS production in normal hearts. Increases in the abundance of the TRPC3-Nox2 complex underlie pathological hypertrophy and atrophy. Voluntary exercise can induce physiological hypertrophy under hemodynamic load by reducing the TRPC3-Nox2 complex.

Article Snippet: TRPC3 was detected with rabbit anti-TRPC3 antibody (1:1,000, ACC-016, Alomone).

Techniques: Activity Assay, Expressing

Journal: Animals : an Open Access Journal from MDPI

Article Title: Age-Related Changes in the Primary Motor Cortex of Newborn to Adult Domestic Pig Sus scrofa domesticus

doi: 10.3390/ani11072019

Figure Lengend Snippet: Primary Antibodies.

Article Snippet: Immunoperoxidase reaction was performed on serial paraffin sections (5 μm) using a rabbit polyclonal anti-CR (1:100, Abcam, ab702) and a mouse monoclonal anti-PV (1:2000, Sigma-Aldrich, P3088, Clone PARV-19) (details reported in ).

Techniques: Produced, Purification