anti appropriate fluorescent secondary  (Cell Signaling Technology Inc)

Journal: Cells

Article Title: Deletion of the Murine Ortholog of the Human 9p21.3 Locus Leads to Insulin Resistance and Obesity in Hypercholesterolemic Mice

doi: 10.3390/cells13110983

Figure Lengend Snippet: Chr4 Δ70/Δ70 does not affect pancreatic β-cell number but may reduce the cell proliferation rate already in young Ldlr −/− ApoB 100/100 mice. ( A ) Pancreatic islet size, ( B ) proportion of α- and β-cells, and ( C ) proliferation of mixed and ( D ) >10,000 μm 2 pancreatic islets in young Ldlr −/− ApoB 100/100 ( n = 5–7) and Chr4 Δ70/Δ70 mice ( n = 5–7). Primary antibody against glucagon was used as a marker for the pancreatic α-cells, and insulin ab was used for the β-cells. For visualization, fluorescent secondary antibodies were used. In the representative figures, glucagon positive cells appear in red and insulin in green. Nuclei were counterstained with DAPI (blue). Ki-67 antibody was used as a marker of cell proliferation and visualized with DAB. ( E ) For the islet function, insulin secretion of Ldlr −/− ApoB 100/100 ( n = 4) and Chr4 Δ70/Δ70 mice ( n = 4) was measured both in fasted state (0 min) and in response to 1 g/kg i.p. glucose at time points 15, 30, 60 and 90 min after the administration. Asterisk (*) indicates statistical significance. Difference in mean between Ldlr −/− ApoB 100/100 and Chr4 Δ70/Δ70 mice was measured by using t -test, and it was considered statistically significant when p < 0.05.

Article Snippet: Pancreatic α- and β-cells were immunostained with primary antibodies against glucagon (Dako A0565, Rabbit anti-human glucagon, Agilent, Santa Clara, CA, USA) and insulin (Dako A0564, Guinea pig anti-insulin, Agilent, Santa Clara, CA, USA) and appropriate fluorescent secondary antibodies (for glucagon, A21442, chicken anti-rabbit A594, Thermo Fisher Scientific, Waltham, MA, USA, and for insulin, BA-7000 Goat anti-guinea pig with A-2011 Fluorescein Avidin DCS, Vector laboratories, Newark, CA, USA).

Techniques: Marker

Journal: Journal of neuroinflammation

Article Title: A systems pharmacology-based approach to identify novel Kv1.3 channel-dependent mechanisms in microglial activation.

doi: 10.1186/s12974-017-0906-6

Figure Lengend Snippet: Fig. 4 Kv1.3 channels regulate microglial taxis and formation of F-actin complexes induced by LPS. a In the gap closure assay of microglial taxis, BV2 cells were grown to near confluence followed by placement of a uniform scratch using a 200-μm pipette tip. Ability of microglia to close this gap was assessed by measuring the percentage of gap closure over a 24-h period. Paired (0 and 24 h) representative images from each treatment group are shown (a, left), and the comparison of percentage gap closure (over 24 h) is shown (a, right) (six replicates per condition). b BV2 microglial transmigration across a transwell membrane (8-μm pore diameter) after exposure to control, ShK-223, ShK-186, LPS or LPS+ShK-223, or ShK-186, towards serum-containing medium (10% fetal bovine serum). Following 24 h of transmigration, cells were detached from undersurface of the insert (0.25% Trypsin), and cells that successfully migrated across the membrane were counted on a hemocytometer (n = 3, independent experiments). c Comparison of F-actin containing focal adhesion complexes in BV2 microglia following exposure to control, ShK-223, LPS, or LPS+ShK-223. Fixed and permeabilized BV2 cells were labeled with phalloidin-rhodamine to detect F-actin (left: immunofluorescence images). The number of focal complexes were counted per cell at ×40 magnification (right) and compared (>25 cells counted per condition). d DCFDA assay of ROS production by brain mononuclear cells isolated from C57BL/6 mice treated with PBS, LPS, ShK-223, or LPS+ShK-223 IP for four consecutive days (n = 3, mice/group). Cells were loaded with DCFDA for 30 min and assayed for ROS activity by flow cytometry. e Flow cytometric phagocytosis assay of fluorescent (PE) microbeads by brain mononuclear cells isolated from C57B6/L mice treated with PBS, LPS, ShK-223, or LPS+ShK-223, n = 3/group). Dotted line: fluorescence of cells not exposed to beads; gray histogram: PBS-treated; black histogram: LPS-treated. The proportions of all phagocytic cells and highly phagocytic cells were compared across treatment groups (*p < 0.05, **p < 0.01, ***p < 0.005)

Article Snippet: The immunoblots were incubated with anti-Kv1.3 rabbit polyclonal antibody (APC 101, Alomone labs, 1:1000) and anti-beta-actin mAb (Cell Signaling, #3873,1:1000) for 24 h and subsequently appropriate fluorescent secondary antibodies (anti-mouse IgG IRDye® 800 conjugate, Rockland, 1:20,000 and antirabbit IgG Alexa Fluor® 680 conjugate, Invitrogen) were added for 1 h. An Odyssey Scanner (LI-COR) was used to visualize labeled proteins.

Techniques: Transferring, Comparison, Transmigration Assay, Membrane, Control, Labeling, Immunofluorescence, Isolation, Activity Assay, Flow Cytometry, Phagocytosis Assay, Fluorescence