Journal: Urology

Article Title: Preliminary immunohistochemical characterization of a monoclonal antibody (pro:4-216) prepared from human prostate cancer nuclear matrix proteins

doi: 10.1016/s0090-4295(97)00337-3

Figure Lengend Snippet: FIGURE 2. NMP composition of normal human prostate (A) and human prostate cancer (C) taken from a radical prostatectomy specimen from a patient with clinical/y localized prostate cancer. High resolution two-dimensional gel electrophoresis of NMP preparations were isolated and analyzed as described in Methods. Panels B and D represent Western immunoblots using PRO:4-2 16 monoclonal antibody; immunoblotting technique as described in Methods. Arrowhead represents the localization of NMP(s) with immunoreactivity to the PRO:4-2 16 mouse monoclonal anti- body generated against human prostate cancer nuclear matrix preparations. kD = molecular weight in thousands; SDS-PAGE = sodium dodecyl sulfate-polyacrylamide gel electrophoresis; pl = isoelectric point.

Article Snippet: The mouse monoclonal antibodies were UROLOGY 50 (51, 1997 801 then detected with a CY3 conjugated goat anti-mouse antibody (Jackson ImmunoResearch, West Grove, Pa) and DNA was visualized with DAPI (4,6-diamidino-2-phenylindole) at 2 /.

Techniques: Two-Dimensional Gel Electrophoresis, Electrophoresis, Isolation, Western Blot, Generated, Molecular Weight, SDS Page, Polyacrylamide Gel Electrophoresis

Journal: Urology

Article Title: Preliminary immunohistochemical characterization of a monoclonal antibody (pro:4-216) prepared from human prostate cancer nuclear matrix proteins

doi: 10.1016/s0090-4295(97)00337-3

Figure Lengend Snippet: FIGURE 3. lmmunohistochemical staining of human prostate tissue with the mouse monoclonal antibody PRO:4- 2 16. Anti-mouse IgM secondary antibody (Vector’s ABC Elite Kit). 3-3’-Diaminobenzidine tetrahydrochloride (DAB) was used as a chromogen following ethyl green counterstain. (A) Histologically normal prostate tissue (nuclei dem- onstrate an IHC intensity score of 0). (B) Histologically normal prostate tissue with the emphasis on stromal nuclei (weakly positively staining stromal nuclei represent an IHC intensity score of 1). (Cl Adenocarcinoma of the prostate from a tumor with Gleason grade 3 + 3 = 6 (IHC intensity score of 3). The cancerous glands surround a noncancerous gland. (0) A prostatic gland demonstrating PIN (positively staining nuclei represent an IHC intensity score of 1 to 2). The arrow in panel B identifies an area of prostatic stroma. The small arrows in panel C identify prostatic ade- nocarcinoma glands and the large arrow in panel C identifies a histologically normal gland found among cancerous glands. The arrow in panel D represents histologic PIN.

Article Snippet: The mouse monoclonal antibodies were UROLOGY 50 (51, 1997 801 then detected with a CY3 conjugated goat anti-mouse antibody (Jackson ImmunoResearch, West Grove, Pa) and DNA was visualized with DAPI (4,6-diamidino-2-phenylindole) at 2 /.

Techniques: Staining

Journal: PloS one

Article Title: Down-regulation of the canonical Wnt β-catenin pathway in the airway epithelium of healthy smokers and smokers with COPD.

doi: 10.1371/journal.pone.0014793

Figure Lengend Snippet: Figure 5. Immunofluorescent assessment of SFRP2 expression in cytospin preparations of brushed small airway epithelium. Small airway epithelial cell cytopreparations of healthy nonsmokers, healthy smokers and smokers with COPD were stained with anti-SFRP2 followed by a Cy3 conjugated secondary antibody (shown in red). Nuclei were stained with DAPI (shown in blue) A–D. Healthy nonsmokers. A. IgG control; B–D. Examples of anti-SFRP2. E–H. Healthy smokers. E. IgG control; F–H. Examples of anti-SFRP2. I–L. Smokers with COPD; I. IgG Control. J–L. Examples of anti-SFRP2. Bar = 10 mm. doi:10.1371/journal.pone.0014793.g005

Article Snippet: Following incubation with the primary antibodies, goat anti-rabbit Cy5 (Jackson ImmunoResearch) was used as a secondary antibody for SFPR2 and goat antimouse Cy3 (Jackson ImmunoResearch) was used as a secondary antibody for b-tubulin IV, mucin 5AC and chromogranin A.

Techniques: Expressing, Staining, Control

Journal: PloS one

Article Title: Down-regulation of the canonical Wnt β-catenin pathway in the airway epithelium of healthy smokers and smokers with COPD.

doi: 10.1371/journal.pone.0014793

Figure Lengend Snippet: Figure 6. Immunofluorescent assessment of SFRP2 expression in the endobronchial biopsies from large airway epithelium. Endobronchial biopsies from large airway of healthy nonsmoker, healthy smokers and smokers with COPD were stained with anti-SFRP2 followed by a Cy3 conjugated secondary antibody (shown in red). Nuclei were stained with DAPI (shown in blue). A–D. Healthy nonsmokers. A. IgG control; B–D. Examples of anti-SFRP2. E–H. Healthy smokers. E. IgG control; F–H. Examples of anti-SFRP2. I–L. Smokers with COPD. I. IgG Control, J–L. Examples of anti-SFRP2. Bar = 10 mm. doi:10.1371/journal.pone.0014793.g006

Article Snippet: Following incubation with the primary antibodies, goat anti-rabbit Cy5 (Jackson ImmunoResearch) was used as a secondary antibody for SFPR2 and goat antimouse Cy3 (Jackson ImmunoResearch) was used as a secondary antibody for b-tubulin IV, mucin 5AC and chromogranin A.

Techniques: Expressing, Staining, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Cytotoxic NKG2C+ CD4 T cells target oligodendrocytes in multiple sclerosis.

doi: 10.4049/jimmunol.1202725

Figure Lengend Snippet: FIGURE 1. Activated NKG2C+ CD4 T cells display a cytotoxic profile. (A and B) The expression of CD56, NKG2A, and NKG2C by human MBP-specific CD4 T cell lines was assessed. (A) Representative FACS dot plots gated on living cells for the isotype controls or CD4+CD3+ T cells. (B) The percentage of CD4+CD3+ T cells (top panel) or CD56+CD4+CD3+ T cells (bottom panel) within each individual MBP-specific T cell line expressing NKG2C or NKG2A is illustrated for nine T cell lines obtained from two donors. A greater proportion of MBP-specific CD4 T cells express NKG2C compared with NKG2A. Moreover, most CD56+ MBP-specific CD4 T cells express NKG2C. (C) Representative FACS dot plots of PBMCs gated on CD3+ CD4+ T cells either ex vivo or following a short in vitro activation. Cells were not treated (Nil) or were treated with different stimuli: anti-CD3 + anti-CD28, PHA, IL-2, IL-12, or IL-15. Only PHA activation induced NKG2C expression on CD4 T cells. (D and E) The expression of molecules involved in cytotoxicity was assessed in four distinct MBP-specific CD4 T cell lines and PHA-activated CD4 T cells from three donors. (D) Representative FACS dot plots gated on CD4+CD3+ T cells that were NKG2C+ (top panels) or NKG2C2 (bottom panels). (E) Percentage of NKG2C+ and NKG2C2 PHA-activated CD4 T cells expressing NKG2D, CD56, NKp46, FasL, and lytic enzymes. Data are mean 6 SEM. *p , 0.05, **p , 0.01, NKG2C+ versus NKG2C2, Student t test (n = 3).

Article Snippet: Snap-frozen sections (10 mm thick) were air-dried, fixed in 4% paraformaldehyde for 10 min, and blocked for nonspecific binding for 1 h and then a mouse anti-human NKG2C Ab (20 mg/ml; R&D Systems) was incubated 1 h at room temperature and then overnight at 4 ̊C.

Techniques: Expressing, Ex Vivo, In Vitro, Activation Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Cytotoxic NKG2C+ CD4 T cells target oligodendrocytes in multiple sclerosis.

doi: 10.4049/jimmunol.1202725

Figure Lengend Snippet: FIGURE 2. NKG2C+ CD4 T cells target HLA-E–expressing human oligodendrocytes. (A) The expression of HLA-E was determined by flow cytometry on human oligodendrocytes that were not treated or were cultured in the presence of IFN-g, IL-1b, or IFN-g + IL-1b. Representative graphs of HLA-E expression on Nogo-A–gated oligodendrocytes (left panel). Fold increase in the HLA-E DMFI of oligodendrocytes from three donors (right panel; mean 6 SEM). Treatment with either IFN-g (j p = 0.0623) or IFN-g + IL-1b (*p , 0.05) consistently elevated HLA-E expression by oligodendrocytes. (B and C) Cytotoxic potential of PHA-activated CD4 T cells and MBP-specific CD4 T cells toward IFN-g + IL-1b–treated oligodendrocytes was evaluated using the CD107a assay after a 8-h coculture. (B) Representative dot plots of CD107a expression on PHA-activated CD4 T cells preincubated with an isotype control, an anti-CD94 blocking Ab, or an anti-NKG2D blocking Ab prior to their addition to oligodendrocytes (left panel). Flow cytometry events were gated on either CD3+CD4+ NKG2C+ or CD31CD41 NKG2C2 cells. Data are from PHA-activated CD4 T cells obtained from three to six donors (mean 6 SEM). The percentage of CD107a-expressing cells for effector cells alone or cells preincubated with an isotype control, an anti-CD94 blocking Ab, an anti- NKG2D blocking Ab, or both anti-CD94 and anti-NKG2D blocking Abs (right panel). (C) Oligodendrocytes were incubated in the absence (no effector) or in the presence of PHA-activated CD4 T cells preincubated with either an isotype control (Isotype) or anti-CD94 + anti-NKG2D blocking Abs, fixed, and stained for Nogo-A. Photomicrographs are representative of five fields from killing assays performed with two distinct CD4 T cell donors. Scale bar, 10 mm. (D) Representative dot plots of CD107a expression on MBP-specific CD4 T cells, preincubated with either an isotype control, an anti-CD94 or alone prior to their addition to oligodendrocytes. Flow cytometry events were gated on CD3+CD4+ cells. Data obtained from six MBP-specific CD4 T cell lines generated from two donors are shown (mean 6 SEM). *p , 0.05, **p , 0.01, isotype versus anti-CD94, anti-NKG2D, or anti-CD94 + anti-NKG2D.

Article Snippet: Snap-frozen sections (10 mm thick) were air-dried, fixed in 4% paraformaldehyde for 10 min, and blocked for nonspecific binding for 1 h and then a mouse anti-human NKG2C Ab (20 mg/ml; R&D Systems) was incubated 1 h at room temperature and then overnight at 4 ̊C.

Techniques: Expressing, Cytometry, Cell Culture, Control, Blocking Assay, Flow Cytometry, Incubation, Staining, Generated

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Cytotoxic NKG2C+ CD4 T cells target oligodendrocytes in multiple sclerosis.

doi: 10.4049/jimmunol.1202725

Figure Lengend Snippet: FIGURE 3. NKG2C+ CD4 T cells are more abundant in MS patients and exhibit a highly cytotoxic profile. PBMCs obtained from untreated MS patients (MS) or normal controls (NC) were analyzed ex vivo for immune molecules. (A) Representative dot plots of events gated on living cells for isotype control or stained samples: CD3+CD4+CD82 (CD4 T cells), CD3+CD42CD8+ (CD8 T cells), or CD32CD56+ (NK cells). One typical example of a control and one MS patient are shown. (B) The percentage of NKG2C expression on CD4 T cells, CD8 T cells, and NK cells observed in blood samples obtained from 17 MS patients and 12 normal controls. (C) Representative dot plots of CD3+CD4+CD82 T cells from untreated MS patients gated on NKG2C+ (top panels) or NKG2C2 (bottom panels) cells for the expression of NKG2A, NKG2D, CD56, NKp46, FasL, granzyme B, and perforin. (D) Pool data obtained from four untreated MS patients; results are mean 6 SEM. *p , 0.05, **p , 0.01, ***p , 0.001, NKG2C+ versus NKG2C2, Student t test (n = 4).

Article Snippet: Snap-frozen sections (10 mm thick) were air-dried, fixed in 4% paraformaldehyde for 10 min, and blocked for nonspecific binding for 1 h and then a mouse anti-human NKG2C Ab (20 mg/ml; R&D Systems) was incubated 1 h at room temperature and then overnight at 4 ̊C.

Techniques: Ex Vivo, Control, Staining, Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Cytotoxic NKG2C+ CD4 T cells target oligodendrocytes in multiple sclerosis.

doi: 10.4049/jimmunol.1202725

Figure Lengend Snippet: FIGURE 4. HLA-E and NKG2C are expressed in MS brain tissues. (A) Representative staining of paraffin-embedded sections with Luxol Fast Blue for myelin. Cortex (C) and white matter (WM) are identified; within the white matter, well-demarcated MS lesions (L) are seen, as well as diffuse areas of demyelination and tissue damage, known as dirty appearing white matter (DAWM). Some areas of cortex contain cortical demyelinated lesions (CL). (B) H&E- stained section of the area of DAWM depicted in (A); tissue is rarefied due to loss of myelin, reactive astrocytes (black arrows) are seen throughout the area, and a mononuclear infiltrate (blue arrowheads) is observed in the perivascular space around a blood vessel. Original magnification 3200. (C) The MS brain tissue characterized in (A) and (B) was also immunostained for HLA-E (green), O4 (red), and nuclear stain (blue). Fluorescent microscopy shows the presence of oligodendrocytes (O4+) expressing HLA-E. Orange arrowheads indicate examples of oligodendrocytes expressing HLA-E; white arrows indicate an HLA-E– expressing cell that is not of oligodendrocyte lineage. Areas in white boxes are shown enlarged in the far right column, together with the corresponding isotype controls (original magnification 33500). Data shown are representative of 15 fields from three distinct MS tissue blocks. Scale bars, 25 mm. (D) An acute MS lesion was also immunostained for HLA-E (red), GFAP (green), and nuclear stain (blue). HLA-E+ cells could be detected, but these cells are not astrocytes (GFAP+). White arrows indicate examples of HLA-E–expressing cells that are not of astrocyte lineage. Area in white box is shown enlarged in the far right column, together with the corresponding isotype control. Original magnification 3400. (E) Frozen brain tissues from patients with MS were immunostained for CD4 (green), NKG2C (red), and nucleus (blue). Fluorescent microscopy reveals the presence of CD4 T cells and NKG2C+ cells. Orange arrowheads indicate examples of NKG2C-expressing CD4 T cells, white arrows indicate an example of an NKG2C+ cell that is not a CD4 T cell, and pink arrows indicate examples of CD4 T cells that do not express NKG2C. Isotype controls and enlarged areas in white boxes are shown in the last column (original magnification 32500– 33500). Three distinct fields obtained from two MS brain specimens, representative of four distinct MS brain samples, are shown. Scale bars, 25 mm.

Article Snippet: Snap-frozen sections (10 mm thick) were air-dried, fixed in 4% paraformaldehyde for 10 min, and blocked for nonspecific binding for 1 h and then a mouse anti-human NKG2C Ab (20 mg/ml; R&D Systems) was incubated 1 h at room temperature and then overnight at 4 ̊C.

Techniques: Staining, Microscopy, Expressing, Control