Journal: Neuroscience

Article Title: The expression of Toll-like receptor 4, 7 and co-receptors in neurochemical sub-populations of rat trigeminal ganglion sensory neurons.

doi: 10.1016/j.neuroscience.2015.09.069

Figure Lengend Snippet: Fig. 1. TLR4 and TLR7 expression within the TG and DRG. Multiple neuronal profiles display intracellular granular IR for TLR4 within the TG (A) and DRG (B), arrows show selected examples of positive neurons. A proportion of non-neuronal cells that were morphologically identified as satellite cells also display a positive stain for TLR4 within both TG and DRG, indicated by asterisks (A, B). Multiple neuronal profiles also display positive IR for TLR7 within the TG (C) and DRG (D), arrows show selected examples. All non-neuronal cells within the tissue are negative for TLR7. Scale bar 50 lm.

Article Snippet: Primary antibodies against TLR4 (1:200, Abcam, Cambridge, UK), TLR7 (1:500, Novus Biologicals, Littleton, CO, USA), LPCAT1 (1:200, ProteinTech, Chicago, IL, USA) or LPCAT2 (1:200, Novus Biologicals, Littleton, CO, USA) were pooled with primary antibodies against either NF200 (1:4000, Sigma–Aldrich, Gillingham, UK), TRPV1 (1:200, Neuromics, Edina, MN, USA) or P2X3 (1:1000, Novus Biologicals, Littleton, CO, USA) and incubated with tissue sections for 24 h, with the exception of TLR4 which required a 48-h incubation, in a humidity chamber at 4 C. After washing with PBS, tissue sections were incubated with a combination of species-specific alexafluor (AF) 488 (10 lg/mL, Life Technologies, Carlsbad, CA, USA), AF555 (10 lg/mL, Life Technologies, Carlsbad, CA, USA) or AF594 (10 lg/mL, Jackson ImmunoResearch, West Grove, PA, USA) conjugated secondary antibodies for 3 h in a dark humidity chamber at room temperature.

Techniques: Expressing, Staining

Journal: Neuroscience

Article Title: The expression of Toll-like receptor 4, 7 and co-receptors in neurochemical sub-populations of rat trigeminal ganglion sensory neurons.

doi: 10.1016/j.neuroscience.2015.09.069

Figure Lengend Snippet: Fig. 2. Positive control staining of primary antibodies. Multiple positively stained profiles can be identified for TLR4 (A, B), TLR7 (C, D) and LPCAT2 (E, F) in the spleen and LPCAT1 (G, H) in the lung. Scale bar 15 lm and 50 lm for spleen and lung images, respectively.

Article Snippet: Primary antibodies against TLR4 (1:200, Abcam, Cambridge, UK), TLR7 (1:500, Novus Biologicals, Littleton, CO, USA), LPCAT1 (1:200, ProteinTech, Chicago, IL, USA) or LPCAT2 (1:200, Novus Biologicals, Littleton, CO, USA) were pooled with primary antibodies against either NF200 (1:4000, Sigma–Aldrich, Gillingham, UK), TRPV1 (1:200, Neuromics, Edina, MN, USA) or P2X3 (1:1000, Novus Biologicals, Littleton, CO, USA) and incubated with tissue sections for 24 h, with the exception of TLR4 which required a 48-h incubation, in a humidity chamber at 4 C. After washing with PBS, tissue sections were incubated with a combination of species-specific alexafluor (AF) 488 (10 lg/mL, Life Technologies, Carlsbad, CA, USA), AF555 (10 lg/mL, Life Technologies, Carlsbad, CA, USA) or AF594 (10 lg/mL, Jackson ImmunoResearch, West Grove, PA, USA) conjugated secondary antibodies for 3 h in a dark humidity chamber at room temperature.

Techniques: Positive Control, Staining

Journal: Neuroscience

Article Title: The expression of Toll-like receptor 4, 7 and co-receptors in neurochemical sub-populations of rat trigeminal ganglion sensory neurons.

doi: 10.1016/j.neuroscience.2015.09.069

Figure Lengend Snippet: Fig. 3. TLR expression in red blood cells as a negative control. Phase-contrast microscopy was used to identify red blood cells (A). No positive staining was observed in red blood cells for TLR4 (B) or TLR7 (C) antibodies. Scale bar 50 lm.

Article Snippet: Primary antibodies against TLR4 (1:200, Abcam, Cambridge, UK), TLR7 (1:500, Novus Biologicals, Littleton, CO, USA), LPCAT1 (1:200, ProteinTech, Chicago, IL, USA) or LPCAT2 (1:200, Novus Biologicals, Littleton, CO, USA) were pooled with primary antibodies against either NF200 (1:4000, Sigma–Aldrich, Gillingham, UK), TRPV1 (1:200, Neuromics, Edina, MN, USA) or P2X3 (1:1000, Novus Biologicals, Littleton, CO, USA) and incubated with tissue sections for 24 h, with the exception of TLR4 which required a 48-h incubation, in a humidity chamber at 4 C. After washing with PBS, tissue sections were incubated with a combination of species-specific alexafluor (AF) 488 (10 lg/mL, Life Technologies, Carlsbad, CA, USA), AF555 (10 lg/mL, Life Technologies, Carlsbad, CA, USA) or AF594 (10 lg/mL, Jackson ImmunoResearch, West Grove, PA, USA) conjugated secondary antibodies for 3 h in a dark humidity chamber at room temperature.

Techniques: Expressing, Negative Control, Microscopy, Staining

Journal: Neuroscience

Article Title: The expression of Toll-like receptor 4, 7 and co-receptors in neurochemical sub-populations of rat trigeminal ganglion sensory neurons.

doi: 10.1016/j.neuroscience.2015.09.069

Figure Lengend Snippet: Fig. 6. Fluorescence micrograph of rat TG double labeled for TLR7-IR (green, A, D, G) plus either TRPV1- (red, B), P2X3- (red, E) or NF200-IR (red, H). The combined images (C, F, I) show co-localization of TLR7-IR with neurochemical markers. A substantial proportion of TLR7-IR neurons co-express TRPV1-IR, examples of co-expressing neurons are indicated by filled arrows (A, B) and by asterisks in the combined image (C). Some neurons expressing low levels of TRPV1-IR neurons do not display TLR7-IR, examples indicated by open arrows (A, B, C). A substantial proportion of TLR7-IR neurons also co-express P2X3-IR, examples of co-expressing neurons are indicated by filled arrows (D, E) and by asterisks in the combined image (F). Also however, not all P2X3-IR neurons display TLR7-IR, examples indicated by open arrows (D, E, F). There was minimal co- localization between TLR7-IR and NF200-IR; examples of co-expressing neurons are indicated by filled arrows (G, H) and by asterisks in the combined image (I). The vast majority of NF200-IR neurons did not express TLR7-IR, examples indicated by open arrows (G, H, I). A similar pattern of expression and co-expression was observed in the DRG (images not shown). Scale bar 50 lm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Primary antibodies against TLR4 (1:200, Abcam, Cambridge, UK), TLR7 (1:500, Novus Biologicals, Littleton, CO, USA), LPCAT1 (1:200, ProteinTech, Chicago, IL, USA) or LPCAT2 (1:200, Novus Biologicals, Littleton, CO, USA) were pooled with primary antibodies against either NF200 (1:4000, Sigma–Aldrich, Gillingham, UK), TRPV1 (1:200, Neuromics, Edina, MN, USA) or P2X3 (1:1000, Novus Biologicals, Littleton, CO, USA) and incubated with tissue sections for 24 h, with the exception of TLR4 which required a 48-h incubation, in a humidity chamber at 4 C. After washing with PBS, tissue sections were incubated with a combination of species-specific alexafluor (AF) 488 (10 lg/mL, Life Technologies, Carlsbad, CA, USA), AF555 (10 lg/mL, Life Technologies, Carlsbad, CA, USA) or AF594 (10 lg/mL, Jackson ImmunoResearch, West Grove, PA, USA) conjugated secondary antibodies for 3 h in a dark humidity chamber at room temperature.

Techniques: Fluorescence, Labeling, Expressing

Journal: Journal of Innate Immunity

Article Title: TLRs1-10 Protein Expression in Circulating Human White Blood Cells during Bacterial and COVID-19 Infections

doi: 10.1159/000536593

Figure Lengend Snippet: Box plot representation of intracellular expression of TLR3 and TLR7 in neutrophils in septic shock and COVID-19 patients compared to healthy donors. The results are presented as mean fluorescence intensity (MFI). The main body of the boxplots show the 1st and 3rd quartiles. Horizontal lines in each box represent the median. * p < 0.05, ** p < 0.005.

Article Snippet: Within 3 h, blood cells were stained using 14 monoclonal antibodies coupled to a fluorescent dye for the 10 TLRs, CD14, CD16, and CD19 antigens: CD19-APC (clone SJ25C1, #17-0198-42); TLR1-PE (clone GD2.F4, #12-9911-42); TLR3-PE (clone TLR3.7, #12-9039-82), TLR10-PE (clone 3C10C5, #12-2909-42) from Invitrogen; CD16-APC-H7 (clone 3G8, #560195); CD14-PE-Cy7 (clone Mφ9, #562698); TLR4-BV421 (clone TF901, #564401); CD180-BV421 (clone G28-8, #743624) from BD Biosciences; TLR2-PerCP (clone 383936, #FAB2616C); TLR5-A488(clone 624915, #FAB6704G); TLR7-PerCP (clone 533707, #IC5875C); TLR8-A488 (clone 935166, #IC8999G); TLR9-A405 (clone 26C593R, #IC36583V) from R&D Systems and TLR6- FITC (clone TLR6.127, #ab72362) from Abcam.

Techniques: Expressing, Fluorescence

Journal: Scientific reports

Article Title: COVID-19 plasma exosomes promote proinflammatory immune responses in peripheral blood mononuclear cells.

doi: 10.1038/s41598-022-26457-8

Figure Lengend Snippet: Figure 6. Cytokine production and TLRs in response to plasma exosomes. (a) PBMCs were treated with the TLR3 inhibitor (10 µM) for 30 min or remained untreated, followed by stimulation with plasma exosomes (4 × 109 ml−1) for 16 h. Production of cytokines was determined by flow cytometry gated on CD4+, CD8+, and CD14+. COV exo, COVID-19 plasma exosome treatment; inh/cov, TLR3 inhibitor treated and COVID-19 plasma exosomes stimulated; non-COV, treatment with non-COVID plasma exosomes. *p < 0.05; ns, p > 0.05. ANOVA, equal variants. (b) CD4+ T cells, CD8+ T cells, and CD14+ monocytes were isolated from PBMC using MicroBeads, followed by treatment with plasma exosomes from COVID-19 patients upon admission (COV exo, 4 × 109 ml−1), non-COVID donors (non- COV exo, 4 × 109 ml−1), or poly(I:C) (5 µg ml−1) for 16 h at 37 °C. Expression of TLR3, TLR7, TLR8, and TLR9 was determined by flow cytometry gated on live cells. Isotype antibodies and no-antibody blanks were used in each flow cytometry assay. ctrl, medium only control. Error bars, ± SD; *p < 0.05; ns, p > 0.05; one-way ANOVA. Isotype antibody controls and blank controls were performed in parallel in flow cytometry.

Article Snippet: For flow cytometry, cells were stained using monoclonal antibodies: PE-conjugated IL-6 (Clone MQ2-13A5, BD Biosciences, Franklin Lakes, NJ), APC-conjugated TNF-α (Clone MAb11, BD Biosciences), PE-CF594-conjugated IL-8 (Clone G265-8, BD Biosciences), PE-CF594-conjugated IL-10 (Clone JES3-19F1, BD Biosciences), APC-R700-conjugated IL-17 (Clone N49-653, BD Biosciences), PerCP-Cy5.5-conjugated IFNγ (Clone B27, BD Biosciences), PerCP-Cy5.5-conjugated TGFβ (Clone TW4-9E7, BD Biosciences), PE-conjugated TLR3 (Clone TLR-104, Biolegend, San Diego, CA), PerCPconjugated TLR7 (Clone 533707, R&D systems), FITC anti-human CD288 (TLR8) antibody (Clone 16018A, Biolegend), and APC-conjugated TLR9 (Clone S16D, Biolegend).

Techniques: Clinical Proteomics, Flow Cytometry, Isolation, Expressing, Control