Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Diet-induced aortic valve disease in mice haploinsufficient for the Notch pathway effector RBPJK/CSL.

doi: 10.1161/ATVBAHA.111.227561

Figure Lengend Snippet: Figure 2. Greater leaflet thickness and increased macrophage deposition in RBPKO/ mice fed an HCVD diet. Shown are Masson Trichrome staining (A to F) of aortic valves indicating collagen deposition (arrows) and Mac3 immuno- staining (G to L) of aortic valves demon- strating macrophage infiltration (red; arrows indicate positive immunostaining) in WT mice fed the CC diet (A and G) or the HCVD diet (D and J), Notch1KO/ mice fed the CC diet (B and H) or the HCVD diet (E and K), and RBPKO/ mice fed the CC diet (C and I) or the HCVD diet (F and L). M and N, Mean data of cusp area (M) and percentage of positively stained sections for Mac3 (N) for all groups (n4 per group). One-way ANOVA and LSD post hoc analysis were used to compare differences between groups. Bars with different letters indi- cate significant differences (P0.05) vs all other groups (LSD post hoc analysis). Scale bar0.1 mm.

Article Snippet: Inflammation was detected with anti-Mac3 rat monoclonal antibody (SC19991, Santa Cruz Technol, 1:200).

Techniques: Staining, Immunostaining

Journal: Nature Communications

Article Title: Reactive astrocytes function as phagocytes after brain ischemia via ABCA1-mediated pathway

doi: 10.1038/s41467-017-00037-1

Figure Lengend Snippet: Astrocytes become phagocytic after transient ischemic injury in vivo. a Representative images of the ischemic penumbra showing FJ-labeled and GFAP-immunostained brain sections at 7 days after MCAO ( n = 10 mice). Arrowheads indicate FJ + -degenerating neuronal debris enwrapped by GFAP + astrocytes. Fourteen images per z-stack image (0.49 µm step). b Representative image of GFAP + , Galectin-3 + astrocyte enwrapped NeuN + , FJ + large neuronal debris. Arrowheads indicate FJ + degenerating neuronal debris enwrapped by GFAP + astrocytes. Thirty-four images per z-stack image (0.3 µm step). c Representative images of NeuN + signals in LAMP2 + lysosomes in GFAP + astrocytes ( arrowheads ). d Processes of Galectin-3 and GFAP double-positive astrocytes enwrapping numerous FJ + small debris ( n = 8 mice). The white squares in the left panel image indicate the region of high magnification of shown in the right panel. e Immunoelectron microscopic (iEM) images of GFAP + astrocytes (gold particles: GP; blue ). Left , an image of an astrocyte in the striatum of an intact mouse. Middle , an image of an astrocyte in the ipsilateral striatum at 3 days after MCAO. Arrowheads indicate phagocytic inclusions. Right , high-magnification image of the box shown in the middle panel . Arrow indicates myelin-like structure. N, nucleus; M, mitochondria; in, phagocytic inclusion. f Percentage of GFAP + astrocytes with phagocytic inclusions in total in the intact ( n = 461 cells, 4 mice) and MCAO-treated striatum ( n = 315 cells, 4 mice, ** P = 0.0056, unpaired t -test). Values represent means ± SEM

Article Snippet: The sections were incubated in the following primary antibodies in blocking solution: monoclonal mouse anti-GFAP (1:2000; Millipore, MAB3402), polyclonal rabbit anti-GFAP (1:2000; Millipore, AB5804), monoclonal rat anti-GFAP (1:2000; Invitrogen, 13-0300), monoclonal mouse anti-NeuN (1:100; Millipore, MAB377), polyclonal rabbit anti-NeuN (1:500; Millipore, MABN140), polyclonal rabbit anti-3PGDH (1:1000; gift from Dr. M. Watanabe), polyclonal rabbit anti-ABCA1 (1:1000; Novus Biologicals, NB400-105), polyclonal rabbit anti-MEGF10 (1:200; Millipore, ABC10), polyclonal rabbit anti-GULP1 (1:100; Novus Biologicals, NBP1-84553), monoclonal rat anti-CD31 (1:100; BD Bioscience, 5502740), monoclonal rat anti-LAMP2 (1:250; Millipore, MABC40), monoclonal rat anti-Galectin-3 (1:500; Cederlane, CL8942AP), monoclonal mouse anti-MAP2 (1:1000; Millipore, MAB378), polyclonal rabbit anti-synapsin I (1:500; Millipore, AB1543), polyclonal rabbit anti-PSD95 (1:250; Cell Signaling, 2507), monoclonal mouse anti-GS (1:250; Millipore, MAB302), polyclonal rabbit anti-Iba1 (1:1000; Wako, 019-19741), monoclonal rat anti-CD11b (1:250; Exbio, 12-595), and monoclonal rat anti-CD68 (1:250; Serotec, MCA1957).

Techniques: In Vivo, Labeling

Journal: Nature Communications

Article Title: Reactive astrocytes function as phagocytes after brain ischemia via ABCA1-mediated pathway

doi: 10.1038/s41467-017-00037-1

Figure Lengend Snippet: Spatiotemporal differences in lysosomal protein between astrocytes and microglia. a Representative images of GFAP, 3PGDH, and LAMP2 immunoreactivity in the contralateral striatum ( upper ) and the ischemic penumbra of the ipsilateral striatum ( lower ) at 7 days after MCAO. Arrowheads indicate astrocytes ( n = 10 mice). Forty images per z-stack image (0.47 µm step). b Quantification of LAMP2 immunoreactivity mean intensity in 3PGDH + astrocytes after MCAO ( n = 9, 9, 9, 16, 24, 9, 18, 26, 9, 16, 24 fields, 3, 4 mice, * P < 0.05, *** P < 0.001 vs. contra (corresponding day), # P < 0.05, ## P < 0.01 ipsi (proximal) vs. ipsi (distal), §§§ P < 0.001 vs. ipsi proxymal D7, one-way ANOVA ( P < 0.0001) with Tukey’s multiple comparison test). c Representative images of GFAP, CD68, and Iba1 immunoreactivity in the ischemic core, penumbra, and contralateral striatum at 3 and 14 days after MCAO ( n = 4 mice). High-magnification images are shown in the right panel . Eighteen images per z-stack image (1.0 µm step). d Quantification of CD68 immunoreactivity mean intensity in Iba1 + microglia after MCAO ( n = 8, 8, 10, 8, 4, 4, 12, 11, 10, 10, 8, 8, 8, 8 fields, 4–6 mice, *** P < 0.001 vs. contra (corresponding day), # P < 0.05, ### P < 0.001 vs. ipsi core (corresponding day), §§§ P < 0.001 vs. respective ipsi core, one-way ANOVA ( P < 0.0001) with Tukey’s multiple comparison test). Values represent means ± SEM

Article Snippet: The sections were incubated in the following primary antibodies in blocking solution: monoclonal mouse anti-GFAP (1:2000; Millipore, MAB3402), polyclonal rabbit anti-GFAP (1:2000; Millipore, AB5804), monoclonal rat anti-GFAP (1:2000; Invitrogen, 13-0300), monoclonal mouse anti-NeuN (1:100; Millipore, MAB377), polyclonal rabbit anti-NeuN (1:500; Millipore, MABN140), polyclonal rabbit anti-3PGDH (1:1000; gift from Dr. M. Watanabe), polyclonal rabbit anti-ABCA1 (1:1000; Novus Biologicals, NB400-105), polyclonal rabbit anti-MEGF10 (1:200; Millipore, ABC10), polyclonal rabbit anti-GULP1 (1:100; Novus Biologicals, NBP1-84553), monoclonal rat anti-CD31 (1:100; BD Bioscience, 5502740), monoclonal rat anti-LAMP2 (1:250; Millipore, MABC40), monoclonal rat anti-Galectin-3 (1:500; Cederlane, CL8942AP), monoclonal mouse anti-MAP2 (1:1000; Millipore, MAB378), polyclonal rabbit anti-synapsin I (1:500; Millipore, AB1543), polyclonal rabbit anti-PSD95 (1:250; Cell Signaling, 2507), monoclonal mouse anti-GS (1:250; Millipore, MAB302), polyclonal rabbit anti-Iba1 (1:1000; Wako, 019-19741), monoclonal rat anti-CD11b (1:250; Exbio, 12-595), and monoclonal rat anti-CD68 (1:250; Serotec, MCA1957).

Techniques:

Journal: PLoS ONE

Article Title: Rab39a Interacts with Phosphatidylinositol 3-Kinase and Negatively Regulates Autophagy Induced by Lipopolysaccharide Stimulation in Macrophages

doi: 10.1371/journal.pone.0083324

Figure Lengend Snippet: (A) Immunostaining of Raw264.7 macrophages expressing EGFP-Rab39a with anti-LAMP2 antibody. (B) Representative sequence images from FRAP analysis of EGFP-Rab39a on latex bead-containing phagosomes. The region marked by a broken-line circle was photobleached at 4 sec, and the recovery of fluorescence was monitored. (C) Temporal changes in fluorescence intensities on the bleached phagosomes. The relative intensity was defined as the ratio of fluorescence intensity at each time point to that at 0 sec. Data represent means and standard errors of means (n=10).

Article Snippet: Rat anti-mouse LAMP2 monoclonal antibody (SouthernBiotech), rabbit anti-LC3 polyclonal antibody (MBL), mouse anti-tubulin monoclonal antibody (Sigma-Aldrich), rabbit anti-p62 antibody (MBL), mouse anti-ubiquitin antibody (MBL), rabbit anti-Beclin1 antibody (MBL), rabbit anti-GM130 antibody (MBL), mouse anti-EGFP monoclonal antibody (Clonetech), rat anti-EGFP monoclonal antibody (Nacalai tesque) , mouse anti-c-Myc antibody (Wako), rabbit anti-Vps34 antibody (CST), mouse anti-UVRAG antibody (MBL) and rabbit anti-ATG14L antibody (MBL) were used as primary antibodies.

Techniques: Immunostaining, Expressing, Sequencing, Fluorescence

Journal: The American Journal of Pathology

Article Title: Endosomal/Lysosomal Processing of Gangliosides Affects Neuronal Cholesterol Sequestration in Niemann-Pick Disease Type C

doi: 10.1016/j.ajpath.2011.04.017

Figure Lengend Snippet: Confocal/immunofluorescence analysis of the localization of GM2, GM3, and GM1 gangliosides (red) and cholesterol (blue) relative to LAMP2 (green) within individual neurons of Npc1−/− mice. A: GM2 and LAMP2. B: GM3 and LAMP2. C: GM1 and LAMP2. D: Cholesterol (filipin) and LAMP2. Note that while there is extensive overlap between LAMP2 and these storage materials, there are also examples of ganglioside- and cholesterol-positive vesicles that are LAMP2 negative (indicated by arrows in each panel). Nucleus, n. Calibration bar in D equals 5 μm and applies to all images.

Article Snippet: Anti-lysosomal membrane glycoprotein 2 (LAMP2) mAb (rat IgG2a, cell culture concentrate) (ABL-93-c) was purchased from Developmental Studies Hybridoma Bank at the University of Iowa (Iowa City, IA).

Techniques: Immunofluorescence

Journal:

Article Title: Biogenesis of Leishmania major -Harboring Vacuoles in Murine Dendritic Cells

doi: 10.1128/IAI.74.2.1305-1312.2006

Figure Lengend Snippet: Biogenesis of PV in immature BMDC (A) and FSDC (B) infected with CMFDA- or CMTMR-labeled L. major promastigotes. At 0.5, 1, 4, or 24 h after pulse infection, the cells were fixed, permeabilized, and stained with antibodies and appropriate secondary reagents to examine the association of CD71, CD68, Rab7, LAMP-1, or LAMP-2 with L. major-containing PV by confocal fluorescence microscopy. Freshly prepared BMDC were identified by CD11c labeling. For each time point and each molecule, the percentages of parasite-containing compartments that colocalized with endosomal/lysosomal proteins were determined after 50 to 300 PV were counted. The data are mean values ± standard errors of the means for two to six independent experiments.

Article Snippet: Thereafter, DC were incubated for 45 min at room temperature in a humid chamber with one of the following primary antibodies in saponin-containing blocking solution: monoclonal antibody (MAb) C2, a rat immunoglobulin G1 (IgG1) recognizing mouse CD71 (transferrin receptor) (BD Pharmingen, Heidelberg, Germany), fluorescein isothiocyanate-conjugated rat anti-mouse LAMP-2 MAb (BD Pharmingen), rabbit anti-mouse Rab4 antibodies (Santa Cruz, Santa Cruz, Calif.), rabbit anti-mouse Rab7 antibodies (Santa Cruz), phycoerythrin-conjugated rat anti-mouse LAMP-1 MAb (Santa Cruz), and biotin-conjugated rat anti-mouse CD68 (macrosialin) MAb (Serotec, Oxford, United Kingdom).

Techniques: Infection, Labeling, Staining, Fluorescence, Microscopy

Journal:

Article Title: Biogenesis of Leishmania major -Harboring Vacuoles in Murine Dendritic Cells

doi: 10.1128/IAI.74.2.1305-1312.2006

Figure Lengend Snippet: Biogenesis of PV in immature BMDC (A) and mature BMDC (B) infected with CMFDA- or CMTMR-labeled L. major promastigotes. At 0.5, 1, 2, or 4 h after pulse infection, the cells were fixed, permeabilized, and stained with antibodies and appropriate secondary reagents to examine the association of CD68, LAMP-1, LAMP-2, CD71, Rab7, or Rab4 with L. major-containing PV by confocal fluorescence microscopy. DC were identified by CD11c labeling. For each time point and each molecule, the percentages of parasite-containing compartments that colocalized with endosomal/lysosomal proteins were determined after 100 to 200 PV were counted. The data are mean values ± standard deviations for two independent experiments.

Article Snippet: Thereafter, DC were incubated for 45 min at room temperature in a humid chamber with one of the following primary antibodies in saponin-containing blocking solution: monoclonal antibody (MAb) C2, a rat immunoglobulin G1 (IgG1) recognizing mouse CD71 (transferrin receptor) (BD Pharmingen, Heidelberg, Germany), fluorescein isothiocyanate-conjugated rat anti-mouse LAMP-2 MAb (BD Pharmingen), rabbit anti-mouse Rab4 antibodies (Santa Cruz, Santa Cruz, Calif.), rabbit anti-mouse Rab7 antibodies (Santa Cruz), phycoerythrin-conjugated rat anti-mouse LAMP-1 MAb (Santa Cruz), and biotin-conjugated rat anti-mouse CD68 (macrosialin) MAb (Serotec, Oxford, United Kingdom).

Techniques: Infection, Labeling, Staining, Fluorescence, Microscopy