Journal: STAR Protocols

Article Title: Isolation, expansion, differentiation, and histological processing of human nasal epithelial cells

doi: 10.1016/j.xpro.2021.100782

Figure Lengend Snippet: Histological analysis of ALI tissue (A) Brightfield image of hematoxylin and eosin-stained day 30 ALI tissue displaying a well ciliated epithelial surface and uniform tissue structure. (B) Fluorescent immunohistochemistry on ALI tissue differentiated for 20 days, multiciliated cells visualized using antibodies against the ciliary constituent acetylated tubulin (green, 1:1000 dilution) and the ciliated cell-specific transcription factor FoxJ1 (Red, 1:100 dilution). DAPI (Blue) was used as a nuclear counterstain. (C) Fluorescent immunohistochemistry on day 20 ALI tissue, secretory cells were visualized with antibodies against the secretoglobin Scgb1A1 (Green, 1:300 dilution), mucous producing cells visualized with antibodies against the mucin MUC5AC (Red, 1:500 dilution). (D) Fluorescent immunohistochemistry on day 30 ALI tissue, visualizing the basal progenitor cell population with antibodies against the transcription factor p63 (Green, 1:100 dilution). All secondary antibodies were used at 1:1000 dilution. Scale bars, 110 μm.

Article Snippet: P63 Polyclonal antibody, working dilution 1:100 , Proteintech , Cat#12143-1-AP.

Techniques: Staining, Immunohistochemistry

Journal: STAR Protocols

Article Title: Isolation, expansion, differentiation, and histological processing of human nasal epithelial cells

doi: 10.1016/j.xpro.2021.100782

Figure Lengend Snippet:

Article Snippet: P63 Polyclonal antibody, working dilution 1:100 , Proteintech , Cat#12143-1-AP.

Techniques: Recombinant, Isolation, Sterility, Membrane, Microscopy, Blocking Assay

Journal: Human reproduction (Oxford, England)

Article Title: Imbalance in the expression of the activating type I and the inhibitory type II interleukin 1 receptors in endometriosis.

doi: 10.1093/humrep/dem021

Figure Lengend Snippet: Figure 1. Interleukin 1 receptor types I and II (IL1R1 and IL1R2) immunostaining in ectopic and eutopic endometrial tissues. Sections of endo- metrial tissue from normal women (A and B) and matched endometrial (D and E) and endometriotic (G and H) tissues from women with endo- metriosis were examined for IL1R1 (A, D and G) or IL1R2 (B, E and H) expression (brown staining). Haematoxylin was used for counterstaining (blue staining). In this figure, tissues were from a normal woman and a woman with endometriosis at days 26 and 24 of the menstrual cycle, respectively. Note the intense immunostaining of IL1R1 in normal (A) and endometriosis (D) women-derived endometrial tissues and particularly in endometriositic tissue (G). Note the intense immunostaining for IL1R2 in a serial section from the endometrial tissue of normal woman (B) and the reduced intensity of IL1R2 immunostaining in serial sections from the endometrial (E) and endometriotic (H) tissues of endometriosis patient. No immunostaining was observed in serial sections incubated with an equivalent concentration of mouse (C) or rabbit (F and I) IgGs instead of the primary mouse anti-IL1R2 antibody or the rabbit anti-IL1R1 antibody, respectively (negative controls). Scale bar: 30 mm.

Article Snippet: Controls included recombinant humansoluble (rhs) IL1R1 and IL1R2 (R&D Systems), used as positive controls, incubation with equivalent concentrations of normal goat IgGs and pre-absorption of the primary anti-IL1R1 and anti-IL1R2 antibodies with 5 mg ml21 (0.09 mM) of rhsIL1R1 and 5 mg ml21 (0.11 mM) of rhsIL1R2, respectively.

Techniques: Immunostaining, Expressing, Staining, Derivative Assay, Incubation, Concentration Assay

Journal: Human reproduction (Oxford, England)

Article Title: Imbalance in the expression of the activating type I and the inhibitory type II interleukin 1 receptors in endometriosis.

doi: 10.1093/humrep/dem021

Figure Lengend Snippet: Figure 2. Simultaneous immunofluorescent staining of IL1R1 and IL1R2 in ectopic and eutopic endometrial tissues. IL11R1 and IL1R2 were immunostained as described in Materials and methods, and DAPI (blue) was used for counterstaining. In this figure, tissues were from a normal woman and a woman with endometriosis at day 26 of the menstrual cycle. Note the green fluorescence corresponding to IL1R1 which is intense in normal (A) and endometriosis (D) women-derived endometrial tissues and particularly in matched endometriotic tissue (G). The red fluorescence corresponding to IL1R2 is markedly less intense in endometriotic (H) and matched endometrial (E) tissues from women with endometriosis than in endometrial tissue from normal women (B). Superposition of the green and the red signals shows simultaneous immu- nostaining (yellow) of IL1R1 and IL1R2. Note the predominant green fluorescence in endometriosis women-derived endometrial (F) and endo- metriotic (I) tissues, when compared with normal women-derived endometrial tissue (C) where a yellow colour corresponding to simultaneous expression of IL1R1 and IL1R2 expression could be seen. Scale bars: 30 mm.

Article Snippet: Controls included recombinant humansoluble (rhs) IL1R1 and IL1R2 (R&D Systems), used as positive controls, incubation with equivalent concentrations of normal goat IgGs and pre-absorption of the primary anti-IL1R1 and anti-IL1R2 antibodies with 5 mg ml21 (0.09 mM) of rhsIL1R1 and 5 mg ml21 (0.11 mM) of rhsIL1R2, respectively.

Techniques: Staining, Derivative Assay, Expressing

Journal: Human reproduction (Oxford, England)

Article Title: Imbalance in the expression of the activating type I and the inhibitory type II interleukin 1 receptors in endometriosis.

doi: 10.1093/humrep/dem021

Figure Lengend Snippet: Figure 3. Graphical illustration of IL1R1 and IL1R2 levels. IL1R1 (A) and IL1R2 (B) concentrations (ng mg21 of total proteins) were measured by ELISA in normal endometrial tissues (NE, n ¼ 27) and in endometrial (EE, n ¼ 25) and endometriotic (ET, n ¼ 25) tissues of patients with endometriosis. Endometriotic tissues were also grouped according to the lesion’s type (ET red, n ¼ 10; ET typical, n ¼ 9; ET white, n ¼ 6). Results were expressed as means + SEM. *P , 0.05, **P , 0.01 and **P , 0.001, respectively, when compared with NE. †P , 0.05 and ††P , 0.001, respectively when compared with EE.

Article Snippet: Controls included recombinant humansoluble (rhs) IL1R1 and IL1R2 (R&D Systems), used as positive controls, incubation with equivalent concentrations of normal goat IgGs and pre-absorption of the primary anti-IL1R1 and anti-IL1R2 antibodies with 5 mg ml21 (0.09 mM) of rhsIL1R1 and 5 mg ml21 (0.11 mM) of rhsIL1R2, respectively.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Human reproduction (Oxford, England)

Article Title: Imbalance in the expression of the activating type I and the inhibitory type II interleukin 1 receptors in endometriosis.

doi: 10.1093/humrep/dem021

Figure Lengend Snippet: Figure 4. Western blot analysis of IL1R1 and IL1R2 expression in the endometrial tissue. For IL1R1 (A), western blotting detected a 90 kDa band, which corresponds to the membrane-bound (mb) form of IL1R1, a 55 kDa band corresponding to the soluble (s) form of the receptor and two other bands of 73.3 and 69 kDa which may correspond to degraded IL1R1 (lane 1). Controls included rhsIL1R1 protein (lane 2) and pre-absorption of the anti-IL1R1 antibody with an excess of rhsIL1R1 (lane 3). For IL1R2 (B), western blotting detected a 68 kDa band, which corresponds to mbIL1R2, and a doublet of 45 and 48 kDa MW corresponding to sIL1R2 (lane 2). Controls included rhsIL1R2 protein (lane 1) and pre-absorption of the anti-IL1R2 anti- body with an excess of rhsIL1R2 (lane 3). In this figure, tissues were from normal women at cycle days 22 (A) and 15 (B).

Article Snippet: Controls included recombinant humansoluble (rhs) IL1R1 and IL1R2 (R&D Systems), used as positive controls, incubation with equivalent concentrations of normal goat IgGs and pre-absorption of the primary anti-IL1R1 and anti-IL1R2 antibodies with 5 mg ml21 (0.09 mM) of rhsIL1R1 and 5 mg ml21 (0.11 mM) of rhsIL1R2, respectively.

Techniques: Western Blot, Expressing, Membrane

Journal: Human reproduction (Oxford, England)

Article Title: Imbalance in the expression of the activating type I and the inhibitory type II interleukin 1 receptors in endometriosis.

doi: 10.1093/humrep/dem021

Figure Lengend Snippet: Figure 5. Western blot analysis of IL1R1 and IL1R2 expression in ectopic and eutopic endometrial tissues. (A) Strips were incubated with a goat polyclonal anti-human IL1R1 antibody. (B) Strips were incubated with a goat polyclonal anti-human IL1R2 antibody. Lane 1, endometrium from a normal woman (cycle day 8); lanes 2 and 3, endometrium and endometriotic tissues from a woman with endometriosis (cycle day 7); lane 4, rhsIL1R1 (A) and rhsIL1R2 (B). a-Actin bands demonstrated equal protein loading.

Article Snippet: Controls included recombinant humansoluble (rhs) IL1R1 and IL1R2 (R&D Systems), used as positive controls, incubation with equivalent concentrations of normal goat IgGs and pre-absorption of the primary anti-IL1R1 and anti-IL1R2 antibodies with 5 mg ml21 (0.09 mM) of rhsIL1R1 and 5 mg ml21 (0.11 mM) of rhsIL1R2, respectively.

Techniques: Western Blot, Expressing, Incubation

Journal: Human reproduction (Oxford, England)

Article Title: Imbalance in the expression of the activating type I and the inhibitory type II interleukin 1 receptors in endometriosis.

doi: 10.1093/humrep/dem021

Figure Lengend Snippet: Figure 6. Dual immnunocytofluorescence of IL1R1 and IL1R2 in endometriotic cells following transfection or not with IL1R2 cDNA. Cells were transiently transfected with pcDNA3-IL1R2 or with the control pcDNA3 vector in chamber slides, IL1R1 and IL1R2 were immunostained and DAPI (blue) was used for counterstaining, as described in Materials and methods. Note the intense green fluorescence corresponding to IL1R1 in endometriotic cells, which were not transfected (A) or transfected with either pcDNA3 (D) or pcDNA3-IL1R2 (G). The red fluorescence corre- sponding to IL1R2 was faint in non-transfected endometriotic cells (B) and in those transfected with pcDNA3 (E) and noticeably more intense in endometriotic cells transfected with pcDNA3-IL1R2 (H). Merging the green and the red signals shows simultaneous immunostaining of IL1R1 and IL1R2 with a predominant green fluorescence in non-transfected (C) or pcDNA3-transfectd cells (F), compared with cells transfected with pcDNA3-IL1R2 (I). Scale bars: 30 mm.

Article Snippet: Controls included recombinant humansoluble (rhs) IL1R1 and IL1R2 (R&D Systems), used as positive controls, incubation with equivalent concentrations of normal goat IgGs and pre-absorption of the primary anti-IL1R1 and anti-IL1R2 antibodies with 5 mg ml21 (0.09 mM) of rhsIL1R1 and 5 mg ml21 (0.11 mM) of rhsIL1R2, respectively.

Techniques: Transfection, Control, Plasmid Preparation, Immunostaining

Journal: Journal of Inflammation Research

Article Title: Screening of Diagnostic Biomarkers and Immune Infiltration Characteristics Linking Rheumatoid Arthritis and Rosacea Based on Bioinformatics Analysis

doi: 10.2147/JIR.S467760

Figure Lengend Snippet: The correlation between infiltrated immune cells and overlapped hub genes. ( A ) The heatmap showed the correlation between infiltrated immune cells and overlapped hub genes in RA. The darker the color, the stronger the correlation. Blue graph represents negative correlation, whereas red graph represents positive correlation between infiltrated immune cells and overlapped hub genes. ( B ) The babble plot showed correlation coefficients ( R ) among each kind of infiltrated immune cells and every single overlapped hub gene in RA. ( C ) The heatmap showed the correlation between infiltrated immune cells and overlapped hub genes in rosacea. The darker the color, the stronger the correlation. Blue graph represents negative correlation, whereas red graph represents positive correlation between infiltrated immune cells and overlapped hub genes. ( D ) The babble plot showed correlation coefficients ( R ) among each kind of infiltrated immune cells and every single overlapped hub gene in rosacea. ( E ) The correlation between CXCL10 and M1 macrophages in RA. ( F ) The expression level of CXCL10 in RA and control. ( G ) The correlation between CCL27 and dendritic cells resting in rosacea. ( H ) The expression level of CCL27 in rosacea and control. Data are presented as the mean ± standard error of the mean. Statistical significance (Student’s t -test): *, p < 0.05; **, p < 0.01; ***, p < 0.001. NC, normal controls.

Article Snippet: Macrophages were treated with recombinant human (rHs) CXCL10 (MedChemExpress, HY-P700533) (0 ng/mL, 2 ng/mL) for 24 h. DC cells were treated with recombinant human (rHs) CCL27 (MedChemExpress, HY-P7767) (0 ng/mL, 2 ng/mL) for 24 h. Both of macrophages and DC cells were collected and used to isolated RNA to detect relative genes expressions by using quantitative real-time polymerase chain reaction (qRT-PCR).

Techniques: Expressing, Control

Journal: Journal of Inflammation Research

Article Title: Screening of Diagnostic Biomarkers and Immune Infiltration Characteristics Linking Rheumatoid Arthritis and Rosacea Based on Bioinformatics Analysis

doi: 10.2147/JIR.S467760

Figure Lengend Snippet: The validation of infiltrated immune cells and diagnostic biomarkers. ( A ) The diagnostic effectiveness of CXCL10 for RA by ROC analysis (left), and the serum CXCL10 concentration in patients of RA (right). ( B ) The diagnostic effectiveness of CCL27 for RA by ROC analysis (left), and the serum CCL27 concentration in patients of rosacea (right). ( C ) The gating strategy for identifying M1 macrophages and M2 macrophages from CD45 + cells. ( D ) The quantitative analysis of the proportions of M1 and M2 macrophages in control and patients with RA (n=6). ( E ) The gating strategy for identifying DC cells from CD45 + cells. ( F ) The quantitative analysis of the proportions of DC cells in control and patients (n=6) with rosacea. ( G ) The mRNA expression levels of TNF-α, IL-6, IL-1β, and IFN-α in macrophages separated from patients with RA or normal control (NC) after CXCL10 treatment for 24h. ( H ) The mRNA expression levels of TNF-α, IL-6, IL-1β, and IFN-α in DC cells separated from patients with rosacea or normal control (NC) after CCL27 treatment for 24h. Data are presented as the mean ± standard error of the mean. Statistical significance (Student’s t -test): **p < 0.01, ***p < 0.001. NC, normal controls.

Article Snippet: Macrophages were treated with recombinant human (rHs) CXCL10 (MedChemExpress, HY-P700533) (0 ng/mL, 2 ng/mL) for 24 h. DC cells were treated with recombinant human (rHs) CCL27 (MedChemExpress, HY-P7767) (0 ng/mL, 2 ng/mL) for 24 h. Both of macrophages and DC cells were collected and used to isolated RNA to detect relative genes expressions by using quantitative real-time polymerase chain reaction (qRT-PCR).

Techniques: Biomarker Discovery, Diagnostic Assay, Concentration Assay, Control, Expressing