Journal: iScience

Article Title: Evidence against the role of toll-like receptors 7 and 8 in sex selection in mice, cattle, and humans

doi: 10.1016/j.isci.2025.113164

Figure Lengend Snippet: Effects of TLR7/8 ligand (R848) on murine sperm partitioning and In vitro fertilization (IVF) (A) The schematic diagram of R848 treatment and swim-up layer assignment. (B) The percentages of sperm in different layers after treatment with R848 at various concentrations, ns = not significant ( p > 0.05). (C) The changes in the percentages of sperm of the same individual mice before and after R848 treatment in each swim-up layer. Data in (B and C) were analyzed using one-way ANOVA. (D and E) Cleavage (D) and blastocyst (E) rates of embryos derived from R848-treated sperm of different layers. Data were analyzed with one-way ANOVA followed by Tukey’s post-hoc tests. ∗ p ≤ 0.05, ∗∗ p ≤ 0.01. (F) The percentages of male embryos derived from IVF using sperm treated with 0 or 0.03 μM R848. The expected male embryo ratio was 50% (red bar). All values are mean ± SD of at least three replicates. (G) Representative agarose gel images of embryo sex determination by PCR. The product sizes for SRY and XIST were 105 and 147 bp, respectively. The bands below the specific ones were likely primer-dimers. Each lane represented an individual blastocyst. Lane marked with “F” and “M” were deemed to be female and male embryos, respectively.

Article Snippet: TLR7 polyclonal antibody , Bioss , Cat# bs-6601R; RRID:AB_11090775.

Techniques: In Vitro, Derivative Assay, Agarose Gel Electrophoresis

Journal: iScience

Article Title: Evidence against the role of toll-like receptors 7 and 8 in sex selection in mice, cattle, and humans

doi: 10.1016/j.isci.2025.113164

Figure Lengend Snippet: TLR7/8 ligand (R848) treatment did not separate X- and Y- sperm (A) Validation of TaqMan real-time PCR for sex chromosome ratio determination by using different ratios of male vs. female mouse genomic DNA. The expected ratios are marked with red bars. Data were analyzed with one sample t test. (B) TaqMan real-time PCR determination of the X-sperm ratios in different layers of sperm treated with R848. Data were analyzed with one-way ANOVA, ns = not significant ( p > 0.05). (C) Schematic diagram of centrifugation of sperm before R848 treatment and swim-up. (D) TaqMan real-time PCR determination of the X-sperm ratios in different layers of sperm from (C). Data were analyzed with one-way ANOVA. All values are mean ± SD from at least three replicates.

Article Snippet: TLR7 polyclonal antibody , Bioss , Cat# bs-6601R; RRID:AB_11090775.

Techniques: Biomarker Discovery, Real-time Polymerase Chain Reaction, Centrifugation

Journal: iScience

Article Title: Evidence against the role of toll-like receptors 7 and 8 in sex selection in mice, cattle, and humans

doi: 10.1016/j.isci.2025.113164

Figure Lengend Snippet: Localization of TLR7/8 in murine sperm (A) Immunofluorescence staining of TLR7/8 (green), acetyl α-tubulin (green), and DNA (blue) in murine caudal epididymal sperm. (i) The Bioss antibody against TLR7 stained the whole sperm tails; (ii) The Abcam antibody against TLR7 stained the lower half of the sperm tail as well as the acrosome of the sperm (red arrow); (iii) TLR8 antibody stained either the lower half (yellow arrow) or the entire sperm tail (blue arrow), as well as the acrosome (red arrow). Scale bar, 25 μm. (B) The percentages of murine sperm stained positively for TLR7/8 by immunofluorescence. (C) Flow cytometry analysis of mouse sperm stained with TLR7/8 and DAPI. In controls (left panels) the sperm were probed with the secondary antibody only and provided the cutoff (vertical lines in the upper panels) for stained and unstained sperm. The cells were detected by the FITC-A ( x axis) and DAPI ( y axis) channels. Each dot represents one sperm. The upper panel showed the distribution of the positively and negatively stained sperm, the lower panel displayed the intensity of fluorescence of the sperm ( x axis) and sperm counts ( y axis). The middle and right panels represent sperm stained for TLR7 and TLR8, respectively. Nearly all sperm were stained by both antibodies. (D) The percentages of mouse sperm positively stained for TLR7 or 8 as detected by flow cytometry. All values are mean ± SD of at least three replicates. Data were analyzed using t tests. ns = not significant ( p > 0.05).

Article Snippet: TLR7 polyclonal antibody , Bioss , Cat# bs-6601R; RRID:AB_11090775.

Techniques: Immunofluorescence, Staining, Flow Cytometry, Fluorescence

Journal: iScience

Article Title: Evidence against the role of toll-like receptors 7 and 8 in sex selection in mice, cattle, and humans

doi: 10.1016/j.isci.2025.113164

Figure Lengend Snippet: Detection of TLR7/8 on bovine X- and Y-sorted sperm (A) Immunofluorescence of TLR7 (green), TLR8 (green), acetyl α-tubulin (green) and DAPI (blue) on frozen-thawed un-sorted, X- and Y-sorted bovine sperm. (i–ii) Both TLR7 and TLR8 were stained at the acrosomal regions of the bovine sperm heads (red arrows), and tails (yellow arrows). Scale bar, 21 μm. (B, C, and E) (B) The percentages of positively stained bovine sperm for TLR7 and TLR8. Western blot analyses of TLR7 (C) and TLR8 (E) on bovine un-sorted (U), X-sorted (X) and Y-sorted (Y) sperm. TLR7 and TLR8 were resolved and blotted using non-reduced and reduced samples, respectively. (D and F) Quantification of the TLR7 (D) and TLR8 (F) bands from western blots. Data were analyzed using one-way ANOVA, ns = not significant ( p > 0.05). (G) Flow cytometry analysis of bovine sperm stained for TLR7/8 and DAPI. In controls (left panels) the sperm were probed with the secondary antibody only, which provided the cutoff (vertical line) for stained and unstained sperm. The cells were detected by the FITC-A ( x axis) and DAPI ( y axis) channels. Each dot represents one sperm. The middle and right panels represent sperm stained for TLR7 and TLR8, respectively. Nearly all sperm were stained by both antibodies. (H) The percentages of positively stained bovine sperm as detected by flow cytometry. Data were analyzed using one-way ANOVA. All values are mean ± SD of at least three replicates, ns = not significant ( p > 0.05).

Article Snippet: TLR7 polyclonal antibody , Bioss , Cat# bs-6601R; RRID:AB_11090775.

Techniques: Immunofluorescence, Staining, Western Blot, Flow Cytometry

Journal: iScience

Article Title: Evidence against the role of toll-like receptors 7 and 8 in sex selection in mice, cattle, and humans

doi: 10.1016/j.isci.2025.113164

Figure Lengend Snippet: Localization of TLR7 and TLR8 on human sperm (A) Representative images of immunofluorescence of TLR7/8 (green) and DAPI (blue) on human sperm. (i–ii) Both TLR7 and TLR8 stained human sperm tails as well as the equator regions of the heads (acrosomes, red arrows). TLR7 was also seen in the head-tail connecting apparatus (Yellow arrow). Scale bar, 22 μm. (B) The percentages of positively stained human sperm by TLR7/8 immunofluorescence. All values are mean ± SD of at least three replicates. Data were analyzed using t tests, ns = not significant ( p > 0.05). (C) Y chromosome fluorescence in situ hybridization (FISH, orange) and immunofluorescence for TLR7 and TLR8 (green) in a human sperm donor because his staining patterns and percentages were different from other donors. The inset (i) shows the details of TLR7 tail stain and the Y chromosome signal. TLR8 stained few sperm and the stain was seen on the entire sperm (ii). Immunofluorescence staining preceded FISH. Scale bar, 25 μm.

Article Snippet: TLR7 polyclonal antibody , Bioss , Cat# bs-6601R; RRID:AB_11090775.

Techniques: Immunofluorescence, Staining, Fluorescence, In Situ Hybridization

Journal:

Article Title: A distinct Toll-like receptor repertoire in human tonsillar B cells, directly activated by Pam 3 CSK 4 , R-837 and CpG-2006 stimulation

doi: 10.1111/j.1365-2567.2006.02392.x

Figure Lengend Snippet: Flow cytometry analysis of Toll-like receptors (TLRs) (open histograms) in freshly isolated human tonsillar CD19+ B cells. Cells were stained intracellularly with mouse monoclonal antibodies (mAbs) against CD19 (J4.119, IgG1), TLR1 (GD2.F4, IgG1), TLR2 (TL2.1, IgG2a) and TLR9 (26C593, IgG1), polyclonal rabbit antibody against TLR7 and appropriate isotype controls (filled histograms). Inserted dot-plots show TLR and CD19. Data from one representative out of four independent experiments are shown. FITC, fluorescein isothiocyanate; PE, phycoerythrin.

Article Snippet: Unlabelled antibodies against TLR1 (GD2.F4) and TLR7 (rabbit, polyclonal) were purchased from Acris antibodies (Hiddenhausen, Germany), and detected using the Alexa Fluor 488 mouse immunoglobulin G1 (IgG1) labelling kit from Molecular Probes (Eugene, OR) or sheep anti-rabbit IgG-PE from Acris.

Techniques: Flow Cytometry, Isolation, Staining

Journal:

Article Title: A distinct Toll-like receptor repertoire in human tonsillar B cells, directly activated by Pam 3 CSK 4 , R-837 and CpG-2006 stimulation

doi: 10.1111/j.1365-2567.2006.02392.x

Figure Lengend Snippet: Tonsil sections stained with antibodies against (a) CD20 (diluted 1 : 1500), (b) CD3 (diluted 1 : 100), (c) Toll-like receptor (TLR)1 (diluted 1 : 50), (d) TLR2 (diluted 1 : 50), (e) TLR7 (diluted 1 : 50) and (f) TLR9 (diluted 1 : 50), were localized using 3,3′-diaminobenzidine (DAB), which stains tissues brown, and analysed by microscopy (magnification ×40–100).

Article Snippet: Unlabelled antibodies against TLR1 (GD2.F4) and TLR7 (rabbit, polyclonal) were purchased from Acris antibodies (Hiddenhausen, Germany), and detected using the Alexa Fluor 488 mouse immunoglobulin G1 (IgG1) labelling kit from Molecular Probes (Eugene, OR) or sheep anti-rabbit IgG-PE from Acris.

Techniques: Staining, Microscopy