Journal:

Article Title: Schistosomal egg antigen-responsive CD8 T-cell population in Schistosoma mansoni -infected BALB/c mice

doi: 10.1046/j.1365-2567.1999.00887.x

Figure Lengend Snippet: Cytokine production after in vitro stimulation of CD8+ T cells from SEACNBr‐immunized mice. CD8+ T cells were isolated from spleens removed 21 days after immunization of BALB/c mice with 50 µg of SEACNBr. IL‐2, IL‐4, IL‐5, IL‐10 and IFN‐γ were measured by specific ELISA in cell‐culture supernatants after 24 hr of stimulation with Con A (10 µg/ml) or anti‐CD3 antibody (5 µg/ml) or 96 hr of stimulation with SEACNBr (50 µg/ml). Results are expressed as mean ± SEM.

Article Snippet: CD8 + T cells were separated by the magnetic cell sorting (MACS) method using colloidal super‐magnetic microbeads conjugated with monoclonal rat anti‐mouse CD8a (Miltenyi Biotec, Bergisch Gladbach, Germany) following the manufacturer's instructions.

Techniques: In Vitro, Isolation, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: Cell transplantation

Article Title: Preemptive Tolerogenic Delivery of Donor Antigens for Permanent Allogeneic Islet Graft Protection

doi: 10.3727/096368914X681027

Figure Lengend Snippet: A. 1×106 to 1×108 ECDI-fixed BALB/c splenocytes (ECDI-SPs) were infused to diabetic B6 recipients on day -7 and day +1. BALB/c islets were transplanted on day 0. Blood glucose (BG) levels were followed until rejection occurred (BG > 250 mg/dl on two consecutive days) or 100 days post transplantation, whichever came first. *p = 0.176, 1×108 vs. 1×107; **p = 0.03, 1×108 vs. 5×106. B. Representative histological examination of rejected grafts (from recipients treated with 5x106 ECDI-SPs) and protected grafts (from recipients treated with 1x108 ECDI-SPs) retrieved on day 20 post transplantation. Asterisks (*) mark discernable islets. Top panels: H&E (magnification: x10); middle panels: triple immunofluorescent staining with insulin, CD4 and Dapi (Magnification: x20); lower panels: triple immunofluorescent staining with insulin, CD8 and Dapi (Magnification: x20). Histology is representative of 3 each islet allografts obtained and sectioned from recipients treated with either the reduced dose (5×106) or the full dose (1×108) of ECDI-SPs.

Article Snippet: For visualization of CD4 and CD8 T cells, sections were stained with rat anti-mouse CD4 mAb (1:250, rat IgG2a, κ clone H129.19; BD Biosciences), or rat anti-mouse CD8 (1:250, rat IgG2a, κ clone 53-6.7; BD Biosciences), followed by biotinylated donkey anti-rat (1:250) and cyanine 3 (Cy3)-streptavidin (BD Biosciences).

Techniques: Transplantation Assay, Staining

Journal: The Journal of Neuroscience

Article Title: Dopamine Transporter Localization in Medial Forebrain Bundle Axons Indicates Its Long-Range Transport Primarily by Membrane Diffusion with a Limited Contribution of Vesicular Traffic on Retromer-Positive Compartments

doi: 10.1523/JNEUROSCI.0744-20.2020

Figure Lengend Snippet: Labeling of HA-DAT in MFB axons by stereotactic injection of HA11 antibodies in vivo. A, Stereotactic injections of HA11 antibodies into the MFB area were performed as described in Materials and Methods. B, Schematics of the experimental protocol for labeling plasma membrane and intracellular HA-DAT. Injected antibody binds to a fraction of plasma membrane HA-DAT and internalizes during animal recovery after injections (∼ 2 h). Animals are then killed, brains are dissected, and sagittal slices are prepared and fixed. Fixed slices are incubated with the secondary A488-conjugated donkey anti-mouse-IgG antibody (αM-A488) at 4°C to label HA11:HA-DAT complexes remaining on the surface of neurons. Following permeabilization with Triton X-100 (TX-100), slices are incubated with rabbit monoclonal anti-HA antibody (RαHA) to label intracellular HA-DAT as well as cell-surface HA-DAT that was not occupied by HA11. In the same incubation, goat polyclonal VPS35 antibody (not shown in the protocol scheme) is included. Secondary donkey anti-rabbit and anti-goat antibodies conjugated with Cy3 and Cy5 are finally used to detect RαHA and VPS35 antibodies, respectively. C, 3D stacks of x-y confocal images were acquired from slices in 35 mm MatTek dishes through 488 nm (green, HA11), 561 nm (red, RαHA), and 640 nm (cyan, VPS35) channels. Maximum intensity projections of three consecutive x-y confocal sections are shown. In the merge A488/Cy3-2 image, A488 fluorescence (HA11, surface HA-DAT) was shifted 4 voxels relative to Cy3 fluorescence (RαHA, total HA-DAT), as shown by an arrow for better visualization of highly similar patterns of A488 and Cy3 fluorescence. Colocalization of 488 nm (plasma membrane HA-DAT) and 561 nm (total HA-DAT) channel fluorescence was also estimated by calculating Pearson correlation coefficient (PCC) using SlideBook6 (n = 6). Insets, High-magnification images of the region indicated by white rectangle in the merge image of HA11, RαHA, and VPS35 fluorescence. Small arrows indicate VPS35 puncta in the axon that do not colocalize with HA-DAT puncta. Large arrow indicates an example of HA-DAT (RαHA) localization in VPS35 containing endosome that lacks HA11. Scale bar, 10 µm.

Article Snippet: Rat IgG2a κ Iso Control was from eBioscience (14-4321-82).

Techniques: Labeling, Injection, In Vivo, Incubation, Fluorescence

Journal: The Journal of Neuroscience

Article Title: Dopamine Transporter Localization in Medial Forebrain Bundle Axons Indicates Its Long-Range Transport Primarily by Membrane Diffusion with a Limited Contribution of Vesicular Traffic on Retromer-Positive Compartments

doi: 10.1523/JNEUROSCI.0744-20.2020

Figure Lengend Snippet: HA-DAT diffusion parameters and internalization in MFB and striatal dopaminergic axons measured in acute sagittal slices. A–D, Acute sagittal brain slices were incubated with the HA11 antibody and Fab fragments of secondary A488-conjugated anti-mouse-IgG antibody at 37°C or room temperature (22°C). Time-lapse images were acquired every 3-5 s at 37°C or 22°C before and after bleaching a small region of an axon. A, Examples of HA-DAT labeling of MFB and striatum in live slices. Scale bars, 10 µm. B, Selected time frames from the time-lapse sequence before and after bleaching of a region of axons in the MFB and striatal areas. Scale bars, 5 µm. C, D, Quantification of the diffusion coefficient (D) and Mf from three independent series of FRAP experiments exemplified in B. Each data point represents calculations of an independent FRAP time-lapse sequence. Differences between D values in MFB and dStr were significant at both 37°C and 22°C (p = 0.012 and p = 0.008, respectively), whereas differences between D values measured at 37°C versus 22°C were not significant. Differences between Mf values were not significant for all variants. One-way ANOVA with Tukey's multiple comparison test was used. E, Internalization assay in acute slices performed under condition recapitulating FRAP measurements at 37°C. Slices were incubated with HA11 antibodies and then Cy3-conjugated Fab fragment of goat anti-mouse Fcγ-specific antibodies at room temperature. After 40 min incubation at 37°C, slices were incubated in nACSF with 5 µg/ml A488-conjugated Fab fragment of goat anti-mouse-IgG antibodies for 30 min at 4°C to label cell-surface HA11:HA-DAT complexes. Slices were then fixed with 4% PFA. F, 3D imaging was performed through 561 nm (red, Cy3; surface plus internalized HA-DAT) and 488 nm (green, A488; surface HA-DAT) channels. Maximum intensity projection images of z stack of 8 consecutive confocal sections are presented. In the Merge2 image, A488 fluorescence was shifted 4 voxels relative to the Cy3 fluorescence as shown by an arrow for better visualization of virtually identical patterns of A488 and Cy3 fluorescence. Insets, High-magnification images of regions indicated by white rectangles. Scale, 10 µm.

Article Snippet: Rat IgG2a κ Iso Control was from eBioscience (14-4321-82).

Techniques: Diffusion-based Assay, Incubation, Labeling, Sequencing, Imaging, Fluorescence

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Characterization of murine non-adherent bone marrow cells leading to recovery of endogenous hematopoiesis

doi: 10.1007/s00018-010-0427-2

Figure Lengend Snippet: Distribution of murine CD4 by real-time PCR and immunohistology. Murine CD4 and isotype control immunohistology staining (streptavidine peroxidase technique) and real-time PCR of gut (day 50). Triple transgenic mice (a,b), C57Bl/6 wild-type mice (c,d), allogeneic NA-BMC transplanted mice (e,f). Shown at 20× original magnification. Quantification of murine CD4 by real-time PCR (g)

Article Snippet: The preparations were incubated with biotin solution (Dako North America) for 10 min, washed with PBS, and incubated with the primary antibody (Rat Anti-Mouse CD4 IgG2aκ; BD Biosciences, San Diego, USA) or isotype control (Rat polyclonal Ig, BD Biosciences, San Diego, USA), diluted 1:100, for 1 h at RT.

Techniques: Real-time Polymerase Chain Reaction, Staining, Transgenic Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Characterization of murine non-adherent bone marrow cells leading to recovery of endogenous hematopoiesis

doi: 10.1007/s00018-010-0427-2

Figure Lengend Snippet: Flow cytometric analysis of murine CD4, murine MHC-I (H2Kd), murine CD8, human CD4 and HLA-DR3 from day 2–61 after transplantation. MuCD4 (a,b), H-2Kd (c), CD8 (d), huCD4 (e), and HLA-DR (f) levels from peripheral blood (gated for lymphocytes) from days 2–61 after co-transplantation of allogeneic bone marrow and allogeneic splenocytes. Results are shown in comparison to transplantation of syngeneic bone marrow cells

Article Snippet: The preparations were incubated with biotin solution (Dako North America) for 10 min, washed with PBS, and incubated with the primary antibody (Rat Anti-Mouse CD4 IgG2aκ; BD Biosciences, San Diego, USA) or isotype control (Rat polyclonal Ig, BD Biosciences, San Diego, USA), diluted 1:100, for 1 h at RT.

Techniques: Transplantation Assay, Comparison