Journal: PLoS ONE

Article Title: The Interaction between Circulating Complement Proteins and Cutaneous Microvascular Endothelial Cells in the Development of Childhood Henoch-Schönlein Purpura

doi: 10.1371/journal.pone.0120411

Figure Lengend Snippet: HMVEC-d were pre-incubated with plasma of patients (N = 30) with acute HSP, plasma of healthy controls (N = 30), or culture medium alone for 48 hr, and then the cells were harvested and analyzed for the expression of C3aR (A) and CD88 (B) by flow cytometry. The expression levels were presented as mean fluorescence intensity (MFI).

Article Snippet: Cells were then harvested by trypsin, washed by PBS, and labeled by PE-conjugated mouse IgG anti-human C3aR, IgG anti-human CD88, or IgG isotype control (BD Biosciences) at 4°C for 30 min. Stained cells were assayed by a FACSCalibur using the CellQuest Software (BD Biosciences).

Techniques: Incubation, Expressing, Flow Cytometry, Fluorescence

Journal: Journal of Immunology Research

Article Title: Protective Effects of Thalidomide on High-Glucose-Induced Podocyte Injury through In Vitro Modulation of Macrophage M1/M2 Differentiation

doi: 10.1155/2020/8263598

Figure Lengend Snippet: Effects of thalidomide on iNOS, CD206, Arg-1 and TNF- α protein expression in 33.3 mM glucose-induced macrophage. (a) iNOS and CD206 protein expressions. (d) Arg-1 protein expression. (f) TNF- α protein expression. The results of iNOS, CD206, Arg-1, and TNF- α were represented in (b), (c), (e), and (g), respectively. All results were expressed as a ration with respect to control and represented as the mean ± SD in triplicates. ∗ p < 0.05; versus 11.1 mM glucose. # p < 0.05; versus 33.3 mM glucose. & p < 0.05; versus Tha100. ^ p < 0.05; versus Tha50. Abbreviations: LPS: lipopolysaccharide; Tha50: 50 μ g/ml thalidomide in 33.3 mM glucose; Tha100: 100 μ g/ml thalidomide in 33.3 mM glucose; Tha200: 200 μ g/ml thalidomide in 33.3 mM glucose; iNOS: inducible nitric oxide synthase; CD206: mannose receptor; TNF- α : tumor necrosis factor- α ; Arg-1: arginase-1.

Article Snippet: After several consecutive rinses with a washing buffer (0.1% Tween-20 in PBS), the membranes were incubated with primary antibodies against iNOS at 1 : 500 dilution (Catalog No. BA0362, Boster), TNF- α (Catalog No. BA0131, Boster) at 1 : 500 dilution, CD206 (Catalog No. A02285-2, Boster) at 1 : 500 dilution, and antibody against Arg-1 (Catalog No. BM4000, Boster) at 1 : 500 dilution overnight at 4°C.

Techniques: Expressing, Control

Journal: Journal of Immunology Research

Article Title: Protective Effects of Thalidomide on High-Glucose-Induced Podocyte Injury through In Vitro Modulation of Macrophage M1/M2 Differentiation

doi: 10.1155/2020/8263598

Figure Lengend Snippet: Effects of thalidomide on iNOS, CD206, Arg-1, and TNF- α mRNA expressions in 33.3 mM glucose-induced macrophage. (a) iNOS mRNA expression. (b) CD206 mRNA expression. (c) CD206 mRNA expression. (d) TNF- α mRNA expression.All the results were represented as the mean ± SD in triplicates ∗ p < 0.05; versus 11.1 mM glucose. # p < 0.05; versus 33.3 mM glucose. & p < 0.05; versus Tha100. ^ p < 0.05; versus Tha50. Abbreviations: LPS: lipopolysaccharide; Tha50: 50 μ g/ml thalidomide in 33.3 mM glucose; Tha100: 100 μ g/ml thalidomide in 33.3 mM glucose; Tha200: 200 μ g/ml thalidomide in 33.3 mM glucose; iNOS: inducible nitric oxide synthase; CD206: mannose receptor; TNF- α : tumor necrosis factor- α ; Arg-1: arginase-1.

Article Snippet: After several consecutive rinses with a washing buffer (0.1% Tween-20 in PBS), the membranes were incubated with primary antibodies against iNOS at 1 : 500 dilution (Catalog No. BA0362, Boster), TNF- α (Catalog No. BA0131, Boster) at 1 : 500 dilution, CD206 (Catalog No. A02285-2, Boster) at 1 : 500 dilution, and antibody against Arg-1 (Catalog No. BM4000, Boster) at 1 : 500 dilution overnight at 4°C.

Techniques: Expressing

Journal: PLoS ONE

Article Title: CX 3 CR1 Is Expressed by Human B Lymphocytes and Meditates CX 3 CL1 Driven Chemotaxis of Tonsil Centrocytes

doi: 10.1371/journal.pone.0008485

Figure Lengend Snippet: ( A ) Flow cytometric analysis of CX 3 CR1 expression in tonsil and blood B cells. Representative histograms from each B cell fractions, and THP-1 and Raji cell lines, tested as positive and negative controls, respectively, are shown. ( B ) Quantization of CX 3 CR1 by real time PCR in peripheral blood and tonsil B cells. Data are normalized to the expression of POLR2A. Values are expressed as arbitrary units calculated as fold of CX 3 CR1 expression relative to the THP-1 cell line, arbitrarily set at 1. Raji cell line was tested as negative control. Data are median, minimum and maximum values from two different experiments performed in quadruplicate. ( C ) Displacement experiments of 125 I-CX 3 CL1 in tonsil and blood B cells. Left panel . Cells were incubated for 2 h at 4°C with 1 nM 125 I-CX 3 CL1 in the absence or presence of 1, 10 and 100 nM cold CX 3 CL1. Percentage of binding inhibition by unlabeled CX 3 CL1, calculated as ratio between cell-bound cpm in the presence of unlabeled ligand and cell-bound cpm in the absence of unlabeled ligand multiplied by 100, was used as a measure for competition between 125 I-labeled and unlabelled CX 3 CL1. THP-1 and Raji cell lines were tested as positive and negative controls, respectively. Right panel . The experiments shown in the left panel were repeated using 100 nM cold CXCL8 as negative control.

Article Snippet: CD10-FITC (MEM78 clone), anti-human IgD-FITC, CD95-FITC, CD44-FITC, anti-Bcl-2-FITC, and anti-Ki-67-FITC mAbs from DAKO (Glostrup, Denmark); unconjugated CD77 and CD39 mAbs from Immunotech, Marseille, France; unconjugated rabbit anti-human CX 3 CR1 (Serotec) that detects an epitope starting from 175 to 189 amino acids; unconjugated CD3, CD56, CD68, CD10, CD27, and anti-human IgM mAbs from DAKO.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Negative Control, Incubation, Binding Assay, Inhibition, Labeling

Journal: PLoS ONE

Article Title: CX 3 CR1 Is Expressed by Human B Lymphocytes and Meditates CX 3 CL1 Driven Chemotaxis of Tonsil Centrocytes

doi: 10.1371/journal.pone.0008485

Figure Lengend Snippet: ( A ) Apoptosis evaluation in purified tonsil GC B cells. The proportion of early apoptotic GC B cells was detected by Annexin V staining at time 0 and after 1, 2, 3, and 4h culture. Results are expressed as median, minimum and maximum values from five different GC B cell suspensions. ( B ) Flow cytometric analysis of CX 3 CR1 expression on freshly purified tonsil GC, naïve, and memory B cells. Results are expressed in box plot as median percent positive cells, minimum and maximum values, and quartiles, from ten different experiments. ( C ) Chemotaxis of GC, naïve, and memory B lymphocytes to rCX 3 CL1. Results are median numbers of migrated cells, maximum and minimum values, from five different experiments for each B cell subset. * P = 0.043 for both 300 and 600 ng/ml rCX 3 CL1. ▪ = Chemotaxis of non-GC B cells to 300 ng/ml rCXCL12 tested as control. ( D ) Freshly isolated GC B cells were pre-incubated with or without PTX and subjected to chemotaxis to 300 ng/ml CX 3 CL1 or medium (nil). Results are median numbers of migrated cells, minimum and maximum values from three different experiments.

Article Snippet: CD10-FITC (MEM78 clone), anti-human IgD-FITC, CD95-FITC, CD44-FITC, anti-Bcl-2-FITC, and anti-Ki-67-FITC mAbs from DAKO (Glostrup, Denmark); unconjugated CD77 and CD39 mAbs from Immunotech, Marseille, France; unconjugated rabbit anti-human CX 3 CR1 (Serotec) that detects an epitope starting from 175 to 189 amino acids; unconjugated CD3, CD56, CD68, CD10, CD27, and anti-human IgM mAbs from DAKO.

Techniques: Purification, Staining, Expressing, Chemotaxis Assay, Control, Isolation, Incubation

Journal: PLoS ONE

Article Title: CX 3 CR1 Is Expressed by Human B Lymphocytes and Meditates CX 3 CL1 Driven Chemotaxis of Tonsil Centrocytes

doi: 10.1371/journal.pone.0008485

Figure Lengend Snippet: ( A ) CX 3 CR1 + and CX 3 CR1 − GC B cells were analyzed after staining with mAbs. Results are percent positive cells, minimum to maximum ranges from ten independent experiments. ( B ) V H 5(D)J H -μ rearrangement sequences were evaluated in CX 3 CR1 + and CX 3 CR1 − GC B cells. 104 molecular clones from five different CX 3 CR1 + GC B cell fractions were compared to 63 molecular clones from three different CX 3 CR1 − GC B lymphocyte fractions. 34 molecular clones from three different CD10 - CD27 − naive B cell fractions were tested as controls. Results are ratios between number of mutations and number of molecular clones.

Article Snippet: CD10-FITC (MEM78 clone), anti-human IgD-FITC, CD95-FITC, CD44-FITC, anti-Bcl-2-FITC, and anti-Ki-67-FITC mAbs from DAKO (Glostrup, Denmark); unconjugated CD77 and CD39 mAbs from Immunotech, Marseille, France; unconjugated rabbit anti-human CX 3 CR1 (Serotec) that detects an epitope starting from 175 to 189 amino acids; unconjugated CD3, CD56, CD68, CD10, CD27, and anti-human IgM mAbs from DAKO.

Techniques: Staining, Clone Assay

Journal: PLoS ONE

Article Title: CX 3 CR1 Is Expressed by Human B Lymphocytes and Meditates CX 3 CL1 Driven Chemotaxis of Tonsil Centrocytes

doi: 10.1371/journal.pone.0008485

Figure Lengend Snippet: ( A ) Double staining of CX 3 CR1 + GC B cells with CD27 and CD23 mAbs. Each B cell fraction was further characterized with mAbs (CD27 + cells, right lower panel; CD23 + cells, left lower panel). Results are median percent positive cells, maximum and minimum values from ten different experiments. ( B ) CX 3 CR1 + GC B cells were subjected to chemotaxis to 300 ng/ml rCX 3 CL1. Migrated cells were double stained for CD27 and CD23. One representative experiment out of seven is shown. ( C ) Purified GC B cells were subjected to CX 3 CL1-driven chemotaxis and migrated cells were collected and stained with a panel of mAbs. Results are median percent positive cells, maximum and minimum values from four different experiments.

Article Snippet: CD10-FITC (MEM78 clone), anti-human IgD-FITC, CD95-FITC, CD44-FITC, anti-Bcl-2-FITC, and anti-Ki-67-FITC mAbs from DAKO (Glostrup, Denmark); unconjugated CD77 and CD39 mAbs from Immunotech, Marseille, France; unconjugated rabbit anti-human CX 3 CR1 (Serotec) that detects an epitope starting from 175 to 189 amino acids; unconjugated CD3, CD56, CD68, CD10, CD27, and anti-human IgM mAbs from DAKO.

Techniques: Double Staining, Chemotaxis Assay, Staining, Purification

Journal: PLoS ONE

Article Title: CX 3 CR1 Is Expressed by Human B Lymphocytes and Meditates CX 3 CL1 Driven Chemotaxis of Tonsil Centrocytes

doi: 10.1371/journal.pone.0008485

Figure Lengend Snippet: ( A ) Splenocytes from WT mice double stained with anti-CX 3 CR1 and B220 mAbs were analyzed by flow cytometry. Histograms from two different WT mice are shown. ( B ) Splenocytes from WT mice were tested for chemotaxis to murine rCX 3 CL1 or rCXCL12 (control). Migrated B cells were enumerated by flow cytometry using anti-B220 mAb. Results are median number of migrated cells, minimum and maximum values from four different experiments. ( C ) CX 3 CR1 − / − , CX 3 CL1 − / − or WT mice were immunized with OVA and tested at different dilutions for specific IgG production by ELISA. Results were expressed as median optical densities (OD), maximum and minimum values, from ten different experiments. Serum from two WT mice was titrated for OVA IgG antibodies (1: 200, 1:500, 1:1000, 1:10000, 1:50000, 1:100000 dilutions) and used in each tests as standard curve, whereas serum from mice before immunization was used as negative control.

Article Snippet: CD10-FITC (MEM78 clone), anti-human IgD-FITC, CD95-FITC, CD44-FITC, anti-Bcl-2-FITC, and anti-Ki-67-FITC mAbs from DAKO (Glostrup, Denmark); unconjugated CD77 and CD39 mAbs from Immunotech, Marseille, France; unconjugated rabbit anti-human CX 3 CR1 (Serotec) that detects an epitope starting from 175 to 189 amino acids; unconjugated CD3, CD56, CD68, CD10, CD27, and anti-human IgM mAbs from DAKO.

Techniques: Staining, Flow Cytometry, Chemotaxis Assay, Control, Enzyme-linked Immunosorbent Assay, Negative Control

Journal:

Article Title: The immunosuppressive surface ligand CD200 augments the metastatic capacity of squamous cell carcinoma

doi: 10.1158/0008-5472.CAN-09-4380

Figure Lengend Snippet: CD200R1+ stromal cells are CD11b+/Gr-1+ MDSCs. A, CD200R1 immunofluorescence in WD SCC, LN Met and Lung Met (white arrows). DAPI counterstain was conducted to visualize nuclei. Dashed line demarcates SCC keratinocytes in LN Met. B, For CD200R1 FACS analysis of CD200R1, CD11b, and Gr-1 or MHC II expression. The total population of CD200R1+ cells were gated and subsequently analyzed for expression of CD11b and Gr-1 or MHC class II. The percentage of the total CD200R1 pool for a single experiment is shown. Far right panel: murine SCC were stained with antibodies against CD200R1 (red) and CD11b (green) and counterstained with DAPI (blue). Arrows point to CD200R1+/CD11b+ (yellow) stromal MDSCs. C, Left: Bar graph showing the densitometric units representing GM-CSF and G-CSF levels in pRS-NS Lung Met/CD200R1+ co-cultures versus pRS-CD200 Lung Met/CD200R1+ co-cultures. *- statistically significant difference (GM-CSF, p = 0.049; G-CSF, p = 0.002). Right: H&E staining (top left) and G-CSF (red) and CD200 (green) immunofluorescence in murine PD SCC. Nuclei were delineated with DAPI (blue). Scale bars mark 50µm.

Article Snippet: Antibodies Antibodies were used against human CD200, mouse CD200, CD3ε, CD86, α6 integrin-FITC (BD Biosciences); NK1.1-488, c-kit, CD123, MHC II, CD11b-FITC, CD11b-PE, Gr-1-FITC, Gr-1-PE, CD11c (BioLegend); Langerin, Foxp3 (eBioscience); Keratin 14 (Covance), CD200R1 (R&D Systems), CD4-APC (Abcam), G-CSF (Santa Cruz), anti-rabbit HRP (Jackson Immunoresearch); species-specific Alexa Fluor-488, -594, -647, and -680 (Invitrogen); and human α6 integrin (kindly provided by Fiona Watt).

Techniques: Immunofluorescence, Expressing, Staining

Journal: The Journal of Neuroscience

Article Title: The Cancer Chemotherapeutic Paclitaxel Increases Human and Rodent Sensory Neuron Responses to TRPV1 by Activation of TLR4

doi: 10.1523/JNEUROSCI.1956-15.2015

Figure Lengend Snippet: A, B, Prevention and reversal of paclitaxel-induced hyperalgesia by intrathecal injection of a TRPV1 antagonist (AMG9810). The baseline (BL) behavioral test in A and B were collected before paclitaxel (Pac) or vehicle (Veh) treatments. In A, the gray shading indicates the time of treatment with 15 μg of AMG9810 (intrathecal) or vehicle solution. In B, paclitaxel-induced mechanical hypersensitivity was confirmed as significant from Veh–Veh-treated rats (open squares, n = 5) at 14 d after treatment (P) in two groups (open and filled circles, n = 5 each); rats were then treated with 15 μg of the TRPV1 antagonist AMG9810 intrathecally (filled circles) or vehicle solution (open circles) as indicated by the arrow. *p < 0.05; **p < 0.01; ***p < 0.001; two-way ANOVA followed by Bonferroni post hoc test. The representative image in C shows the baseline staining of TRPV1 (red) in the DRG in vehicle-treated rats that was not different from naive rats (data not shown); TRPV1 staining becomes elevated by day 7 after paclitaxel treatment (D). Cotreatment of rats with PBS (intrathecally) with paclitaxel did not affect the increased staining of TRPV1 (E), whereas cotreatment with LPS-RS (intrathecally) completely prevented the increase in TRPV1 (F). As indicated by the bar graphs in G, the increase in TRPV1+ neurons by paclitaxel was statistically significant (**p < 0.01), whereas in H, this was significantly less in the LPS-RS-treated rats versus the PBS-treated rats. Scale bar, 100 μm. **p < 0.01.

Article Snippet: After blocking in 5% normal donkey serum and 0.2% Triton X-100 in PBS for 1 h at room temperature, the sections were incubated overnight at 4°C in 1% normal donkey serum and 0.2% Triton X-100 in PBS containing primary antibodies against the following targets: TRPV1 (rabbit anti-rat, 1:1000; Alomone), TLR4 (mouse anti-rat, 1:200; Abcam), IB4 (1:1000 BS-Isolectin B4 FITC Conjugate; Sigma-Aldrich), and CGRP (mouse anti-rat, 1:1000; Abcam).

Techniques: Injection, Staining

Journal: The Journal of Neuroscience

Article Title: The Cancer Chemotherapeutic Paclitaxel Increases Human and Rodent Sensory Neuron Responses to TRPV1 by Activation of TLR4

doi: 10.1523/JNEUROSCI.1956-15.2015

Figure Lengend Snippet: TRPV1 is colocalized with TLR4 in DRG neurons and afferent terminals in the spinal cord. TRPV1 alone is shown in red in the left column for DRG neurons in A and in spinal terminals in D. TLR4 staining in subsets of DRG neurons is shown in green in B and spinal terminals in E (center column). Colocalization of the two are shown in the merged image by yellow for DRG in the right column (C), as well as in fiber profiles in the superficial spinal dorsal horn (F). Scale bar, 100 μm. The representative recording in G shows that acute application of paclitaxel (12.5 μm) evoked spontaneous action potentials in subsets of DRG neurons from animals treated with paclitaxel. In H and I, representative action potential waveforms for the neuron in G evoked by direct current injection after 5 min of vehicle treatment and then after 5 min of acute perfusion with 12.5 μm paclitaxel are shown. The bar graphs in J show the group data for the effects of paclitaxel on several action potential properties. AP, Action potential; RMP, resting membrane potential; AHP, after-hyperpolarization; AHPxx%, interval to each percentage of maximal amplitude. *p < 0.05; **p < 0.01; ***p < 0.001 paclitaxel versus vehicle; paired t test.

Article Snippet: After blocking in 5% normal donkey serum and 0.2% Triton X-100 in PBS for 1 h at room temperature, the sections were incubated overnight at 4°C in 1% normal donkey serum and 0.2% Triton X-100 in PBS containing primary antibodies against the following targets: TRPV1 (rabbit anti-rat, 1:1000; Alomone), TLR4 (mouse anti-rat, 1:200; Abcam), IB4 (1:1000 BS-Isolectin B4 FITC Conjugate; Sigma-Aldrich), and CGRP (mouse anti-rat, 1:1000; Abcam).

Techniques: Staining, Injection

Journal: The Journal of Neuroscience

Article Title: The Cancer Chemotherapeutic Paclitaxel Increases Human and Rodent Sensory Neuron Responses to TRPV1 by Activation of TLR4

doi: 10.1523/JNEUROSCI.1956-15.2015

Figure Lengend Snippet: TRPV1 sensitization by paclitaxel and LPS in DRG neurons shown using calcium imaging. Representative calcium imaging results of change in 340/380 ratio in dissociated DRG neurons after perfusions of capsaicin (CAP, 200 nm) alone and in combination with paclitaxel (12.5 μm) or paclitaxel plus LPS-RS (2 μg/ml) are shown in A–D, each colored line is a single neuron and the time of each application is indicated by the bars over the traces. The bar graphs show the grouped results for experiments testing the interactions between paclitaxel on the responses to capsaicin (E–G) on the effects of LPS on DRG neurons alone (H) and the effects of LPS on DRG neurons from vehicle- and paclitaxel-treated rats (I). **p < 0.01; ***p < 0.001, vehicle rats versus paclitaxel rats; ###p < 0.001, CAP+Pac versus CAP+Pac+LPS-RS.

Article Snippet: After blocking in 5% normal donkey serum and 0.2% Triton X-100 in PBS for 1 h at room temperature, the sections were incubated overnight at 4°C in 1% normal donkey serum and 0.2% Triton X-100 in PBS containing primary antibodies against the following targets: TRPV1 (rabbit anti-rat, 1:1000; Alomone), TLR4 (mouse anti-rat, 1:200; Abcam), IB4 (1:1000 BS-Isolectin B4 FITC Conjugate; Sigma-Aldrich), and CGRP (mouse anti-rat, 1:1000; Abcam).

Techniques: Imaging

Journal: The Journal of Neuroscience

Article Title: The Cancer Chemotherapeutic Paclitaxel Increases Human and Rodent Sensory Neuron Responses to TRPV1 by Activation of TLR4

doi: 10.1523/JNEUROSCI.1956-15.2015

Figure Lengend Snippet: Interactions between TLR4 and TRPV1 in HEK 293 cells studied using whole-cell patch clamp. Inward currents were recorded in HEK293 cells transfected with TLR4 only (top line), TRPV1 only (second line), and both TLR4 and TRPV1 (line 3). The bar graphs at the bottom show the summarized responses with statistical differences determined by paired t tests. Capsaicin (200 nm) did not induce inward currents in cells expressing TLR4 alone, whereas the responses to repeated capsaicin showed desensitization in cells expressing TRPV1 alone. LPS (10 ng/ml) and paclitaxel (12.5 μm) sensitized the responses to capsaicin in cells expressing both TLR4 and TRPV1. *p < 0.05, LPS+capsaicin; ***p < 0.001, paclitaxel+capsaicin; *p < 0.05 vehicle solution+capsaicin versus first capsaicin response.

Article Snippet: After blocking in 5% normal donkey serum and 0.2% Triton X-100 in PBS for 1 h at room temperature, the sections were incubated overnight at 4°C in 1% normal donkey serum and 0.2% Triton X-100 in PBS containing primary antibodies against the following targets: TRPV1 (rabbit anti-rat, 1:1000; Alomone), TLR4 (mouse anti-rat, 1:200; Abcam), IB4 (1:1000 BS-Isolectin B4 FITC Conjugate; Sigma-Aldrich), and CGRP (mouse anti-rat, 1:1000; Abcam).

Techniques: Patch Clamp, Transfection, Expressing

Journal: The Journal of Neuroscience

Article Title: The Cancer Chemotherapeutic Paclitaxel Increases Human and Rodent Sensory Neuron Responses to TRPV1 by Activation of TLR4

doi: 10.1523/JNEUROSCI.1956-15.2015

Figure Lengend Snippet: Representative examples of whole-cell recordings for substantia gelatinosa (SG) neurons before (left) and after (right) administration of the TRPV1 antagonist AMG9810 (5 μm) in the vehicle-treated group (A, B), day 7 paclitaxel-treated group (C, D), and day 14 paclitaxel-treated group (E, F) show increased sEPSCs only in the day 7 paclitaxel-treated group that is suppressed by the AMG9810. The washout segment is not shown. Bar graphs in G and H summarize the mean (±SEM) change in amplitude (G) and frequency (H) of sEPSCs before and after 5 min of AMG9810 application. **p < 0.01, day 7 paclitaxel-treated group versus vehicle and day 14 paclitaxel-treated groups; two-way ANOVA followed by Newman–Keuls post hoc test.

Article Snippet: After blocking in 5% normal donkey serum and 0.2% Triton X-100 in PBS for 1 h at room temperature, the sections were incubated overnight at 4°C in 1% normal donkey serum and 0.2% Triton X-100 in PBS containing primary antibodies against the following targets: TRPV1 (rabbit anti-rat, 1:1000; Alomone), TLR4 (mouse anti-rat, 1:200; Abcam), IB4 (1:1000 BS-Isolectin B4 FITC Conjugate; Sigma-Aldrich), and CGRP (mouse anti-rat, 1:1000; Abcam).

Techniques:

Journal: The Journal of Neuroscience

Article Title: The Cancer Chemotherapeutic Paclitaxel Increases Human and Rodent Sensory Neuron Responses to TRPV1 by Activation of TLR4

doi: 10.1523/JNEUROSCI.1956-15.2015

Figure Lengend Snippet: Paclitaxel application increased mEPSC frequency in superficial dorsal horn neurons in rat spinal cord slice. A, Native recording of mEPSC activity before and after paclitaxel (50 nm) application. B, The TRPV1 antagonist SB366791 (10 μm) did not change the mEPSC frequency but prevented its increase during coapplication with paclitaxel. C, Averaged responses demonstrate that paclitaxel treatment induced a significant increase in mEPSC frequency compared with the baseline (control, 100%) value (140.7 ± 11.1%; n = 14). This increase was prevented by the TRPV1 antagonist (SB366791+paclitaxel) treatment, whereas the antagonist alone had no effect (SB366791; n = 10). D, Paclitaxel (50 nm) application did not change the frequency or amplitude of the sEPSCs or the amplitude of the dorsal root sEPSCs (E). ***p < 0.001 versus control values; ###p < 0.001 versus paclitaxel; one-way ANOVA followed by Student–Newman–Keuls test.

Article Snippet: After blocking in 5% normal donkey serum and 0.2% Triton X-100 in PBS for 1 h at room temperature, the sections were incubated overnight at 4°C in 1% normal donkey serum and 0.2% Triton X-100 in PBS containing primary antibodies against the following targets: TRPV1 (rabbit anti-rat, 1:1000; Alomone), TLR4 (mouse anti-rat, 1:200; Abcam), IB4 (1:1000 BS-Isolectin B4 FITC Conjugate; Sigma-Aldrich), and CGRP (mouse anti-rat, 1:1000; Abcam).

Techniques: Activity Assay

Journal: The Journal of Neuroscience

Article Title: The Cancer Chemotherapeutic Paclitaxel Increases Human and Rodent Sensory Neuron Responses to TRPV1 by Activation of TLR4

doi: 10.1523/JNEUROSCI.1956-15.2015

Figure Lengend Snippet: Immunofluorescent double staining shows that TRPV1 (red, A) is colocalized with TLR4 (green, B) in human DRG neurons (yellow in merged image at right, C). The red arrows in the merged image indicate cells only showing TRPV1, the green arrows indicate cells only expressing TLR4, and the yellows arrow points to cells positive for both TRPV1 and TLR4. Scale bar, 200 μm. Three types of responses were observed when human DRG neurons were tested by application of capsaicin (Cap) and paclitaxel (Pac). Type 1 neurons did not respond to either capsaicin or paclitaxel (data not shown). Type 2 neurons (D) responded positively to capsaicin (left column), showed no responses to paclitaxel (center column), and then showed desensitization to a second application of capsaicin (right column). Type 3 neurons (E) showed responses to capsaicin (left column) and to paclitaxel (center column) and then showed a facilitation of response to the repeated application of capsaicin (right column). The bar graphs at the bottom show the summarized response for the type 2 and 3 neurons. The baseline response to capsaicin was not different between groups. The second response to capsaicin was significantly reduced compared with the first in the type 2 neurons. The type 3 neurons showed significantly greater responses to paclitaxel than did type 2 neurons; and type 3 neurons showed a significantly increased response to the second application of capsaicin compared with the first. *p < 0.05; Mann–Whitney U test.

Article Snippet: After blocking in 5% normal donkey serum and 0.2% Triton X-100 in PBS for 1 h at room temperature, the sections were incubated overnight at 4°C in 1% normal donkey serum and 0.2% Triton X-100 in PBS containing primary antibodies against the following targets: TRPV1 (rabbit anti-rat, 1:1000; Alomone), TLR4 (mouse anti-rat, 1:200; Abcam), IB4 (1:1000 BS-Isolectin B4 FITC Conjugate; Sigma-Aldrich), and CGRP (mouse anti-rat, 1:1000; Abcam).

Techniques: Double Staining, Expressing, MANN-WHITNEY