Journal: Histochemistry and Cell Biology

Article Title: Stage-specific expression of Toll-like receptors in the seminiferous epithelium of mouse testis

doi: 10.1007/s00418-024-02310-z

Figure Lengend Snippet: Expression pattern of TLR-12 in the cycle of seminiferous epithelium. Anti-TLR-12 (NBP2-24,833, Novus, 1/50) primary antibody was used for detection of cells expressing TLR-12. Immune-positive cells expressing TLR-12 ( a , b , c ) appear brown in color. TLR-12 is expressed in spermatocytes, round and elongated spermatids, endosomal compartments and acrosomes ( a , b , c ). Please note that acrosomes of round spermatids ( a , b ), endosomal compartments of both spermatocytes ( b , c ) and elongated spermatids ( c ) are immune positive. Please note that PAS staining on the corresponding sequential section (Supplementary Fig. ) indicates that TLR-12 is expressed at early stage of spermatogenesis. Arrow heads indicate immunopositivity at the acrosome. eSt elongated spermatid, Ec endosomal compartment, rSt round spermatid, Ser Sertoli cell, *spermatocytes, and arrows indicate spermatogonia

Article Snippet: Anti-TLR-1 (B-23, Sc-130896, Santa Cruz Biotechnology, 1/50), anti-TLR 2 (NB100-56720, Novus, 1/50), anti-TLR-3 (NB100-56571, Novus, 1/50), anti-TLR-4 (NB100- 56,566, Novus, 1/50), anti-TLR-5 (H-127, Sc-10742, Santa Cruz Biotechnology, 1/50), anti-TLR-6 (NBP1-54,336, Novus, 1/50), anti-TLR-7 (NB100-56682, Novus, 1/50), anti-TLR-8 (NBP2- 24,917, Novus, 1/50), anti-TLR-9 (NBP2-24,729, Novus, 1/50), anti-TLR-11 (NBP1-77,204, Novus, 1/50), anti-TLR-12 (NBP2-24,833, Novus, 1/50), and anti-TLR-13 (NBP2-24,539, Novus, 1/50) were used as primary antibodies.

Techniques: Expressing, Staining

Journal: Histochemistry and Cell Biology

Article Title: Stage-specific expression of Toll-like receptors in the seminiferous epithelium of mouse testis

doi: 10.1007/s00418-024-02310-z

Figure Lengend Snippet: Expression pattern of TLR-13 in the cycle of seminiferous epithelium. Anti-TLR-13 (NBP2-24,539, Novus, 1/50) primary antibody was used for detection of cells expressing TLR-13. Immune-positive cells expressing TLR-13 appear brown in color. TLR-13 is expressed only in elongated spermatids and residual bodies. Please note that the presence of two immune-positive residual bodies located at the luminal surface of the seminiferous of epithelium (arrows). Please note that PAS staining on the corresponding sequential section (Supplementary Fig. ) indicates that TLR-13 is expressed at early stage of spermatogenesis. eSt elongated spermatid, Rb residual body, rSt round spermatid, *spermatocytes

Article Snippet: Anti-TLR-1 (B-23, Sc-130896, Santa Cruz Biotechnology, 1/50), anti-TLR 2 (NB100-56720, Novus, 1/50), anti-TLR-3 (NB100-56571, Novus, 1/50), anti-TLR-4 (NB100- 56,566, Novus, 1/50), anti-TLR-5 (H-127, Sc-10742, Santa Cruz Biotechnology, 1/50), anti-TLR-6 (NBP1-54,336, Novus, 1/50), anti-TLR-7 (NB100-56682, Novus, 1/50), anti-TLR-8 (NBP2- 24,917, Novus, 1/50), anti-TLR-9 (NBP2-24,729, Novus, 1/50), anti-TLR-11 (NBP1-77,204, Novus, 1/50), anti-TLR-12 (NBP2-24,833, Novus, 1/50), and anti-TLR-13 (NBP2-24,539, Novus, 1/50) were used as primary antibodies.

Techniques: Expressing, Staining

Journal: PLoS Pathogens

Article Title: HIV and HCV Activate the Inflammasome in Monocytes and Macrophages via Endosomal Toll-Like Receptors without Induction of Type 1 Interferon

doi: 10.1371/journal.ppat.1004082

Figure Lengend Snippet: Monocytes were cultured without stimulation or with HIV BaL or HCV Subject 180 and IL-1β, IFNα and measured at multiple timepoints. Fold change in expression of IL-1β (black bar) and IFNα (grey bar) following HIV BaL or HCV Subject 180 relative to stimulation with media alone is shown at 6 h ( A ). Functional knockdowns of the endosomal located TLRs in monocytes were generated by RNA interference. Monocytes were mock transfected or transfected with either siRNA targeting TLR7 or a non-targeting sequence (Scramble). After 24 h, cell lysates were prepared and efficacy of knockdown determined by ( B ) western blot and ( C ) qRT-PCR. ( D ) Specificity of the TLR7 siRNA was confirmed by qRT-PCR using primers for TLR3, TLR7, TLR8, and TLR9. (*) denotes comparisons with p≤0.05 compared to the mock transfected cells. Monocytes in which endosomal TLR knockdown were generated were cultured with HIV BaL (solid bars) or HCV Subject 180 (hatched bars) and pro-IL-1β mRNA transcription measured at 6 h ( E–H ) and IL-18 secretion measured at 24 h ( I–L ). Shown are the relative production of pro-IL-1β mRNA and IL-18 in TLR8 ( E , I ), TLR7 ( F , J ), TLR3 ( G , K ) and TLR9 ( H , L ) knockdown monocytes normalized to mock transfected monocytes (no siRNA) stimulated with the same viruses. Bars represent the mean ± S.D. for n = 6–9 independent transfection experiments, (**) denotes comparisons with p≤0.05 and (***) denoted p≤0.001 compared to the scramble siRNA transfected cells.

Article Snippet: Antibodies anti-Human CD81 mouse mAb (clone 1.3.3.22, Santa Cruz Biotechnology, Santa Cruz CA); Anti-human CD4 mouse mAb (clone SK3) and an isotype control (clone MOPC-21, Biolegend, San Diego CA); Anti-human TLR3 rabbit mAb (clone D10F10, Cell Signaling Technology, Danvers MA); Anti-human TLR7 rabbit pAb (#2633S, Cell signaling Technology); Anti-human TLR8 rabbit mAb (clone D3Z6J, Cell Signaling Technology); Anti-human TLR9 rabbit mAb (clone D2C9, Cell Signaling Technology); Anti-human MyD88 rabbit mAb (clone D80F5, Cell Signaling Technology); Anti-human TRIF/TICAM-1 (clone MAB6216, R&D Systems, Minneapolis MN) mouse mAb; Anti-human NALP3 rabbit pAb (Imgenex, San Diego CA); Anti-human RIG-I (D14G6) rabbit mAb (clone D14G6, Cell Signaling Technology); Anti-human AIM2 rabbit pAb (Abcam, Cambridge, MA) and Anti-human Actin (A2066, Sigma-Aldrich) were purchased from respective vendors.

Techniques: Cell Culture, Expressing, Functional Assay, Generated, Transfection, Sequencing, Knockdown, Western Blot, Quantitative RT-PCR

Journal: Respiratory Research

Article Title: Roflumilast improves corticosteroid resistance COPD bronchial epithelial cells stimulated with toll like receptor 3 agonist

doi: 10.1186/s12931-015-0179-5

Figure Lengend Snippet: Expression of TLR8 in lung tissues of non-smokers, smokers, and COPD patients. Total protein and mRNA were obtained from lung tissues of non-smokers (n = 15), smokers (n = 12), and COPD patients (n = 15). TLR8 protein and mRNA expression were determined by western blot (A) and real time PCR (B) in lung parenchyma. (A) Representative images of western blot for TLR8 and corresponding densitometry expressed as ratio to β-actin. (B) TLR8 mRNA expression given as the ratio to GAPDH. (C, D, E) Lung sections were immunostained for TLR8 and quantified by means of immunohistochemical score of TLR8 in alveolar macrophages (C) and bronchial epithelial cells (D) . (E) Representative immunohistochemistry images are shown. The control IgG isotype showed negative staining. Data are presented as individual values and mean ± standard deviation. Exact P values were obtained using Kruskal-Wallis and Dunn’s post-hoc tests.

Article Snippet: The membrane was blocked with 5% Marvel in PBS containing 0.1% Tween20 (PBS-T), probed with a rabbit anti-human TLR3 polyclonal antibody (1:1000; Bioss, Woburn, USA), rabbit anti-human TLR7 polyclonal antibody (1:1000; Novus Biologicals®, Madrid, Spain), rabbit anti-human TLR8 polyclonal antibody (1:1000; Novus Biologicals®, Madrid, Spain), and normalised to total mouse anti-human β-actin monoclonal antibody (1:1000; Sigma).

Techniques: Expressing, Western Blot, Real-time Polymerase Chain Reaction, Immunohistochemical staining, Immunohistochemistry, Control, Negative Staining, Standard Deviation