Journal: Cell Reports

Article Title: Reconstitution of the Human Nigro-striatal Pathway on-a-Chip Reveals OPA1-Dependent Mitochondrial Defects and Loss of Dopaminergic Synapses

doi: 10.1016/j.celrep.2019.11.111

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-TOMM20 , Novus , NBP1-81556.

Techniques: Recombinant, Virus, Software

Journal: Journal of Cell Science

Article Title: Redefining colocalization analysis with a novel phasor mixing coefficient

doi: 10.1242/jcs.264388

Figure Lengend Snippet: The PMC recapitulates the expected quantification across a wide range of biomolecular association. (A–C) Examples illustrating how the PMC quantifies known high (A), moderate (B) and low (C) biomolecular associations. (A) Color mixing analysis of U2OS cells with mitochondria double-labeled by using an anti-TOMM20 antibody with both AF488-conjugated and AF555-conjugated secondary antibodies. The merged pseudo-color image (left; AF488 in cyan; AF555 in magenta) was extracted from the multispectral image. Scale bar: 10 µm. The phasor plot (middle) exhibits a single narrow cluster near the midpoint of the mixing band. The CMI (right) highlights essentially all mitochondria. The brighter regions near the nucleus reflect a slight difference in depth throughout the cell. (B) Color mixing analysis of a PtK2 cell with MHC labeled with AF488 and phosphorylated RLC labeled with AF568. The merged pseudo-color image (left; AF488 in cyan, AF568 in magenta) was extracted from the multispectral image. Scale bars: 20 µm. The phasor plot (middle) exhibits a single broad cluster centered near the midpoint of the mixing band. The cluster near the origin corresponds to the significant number of background pixels that are excluded from the mixing band. The CMI (right) highlights regions with significant mixing (solid white arrows) and minimal mixing (dashed white arrows). (C) Color mixing analysis of U2OS cells with mitochondria labeled with anti-TOMM20 antibody and an AF488-conjugated secondary antibody, and with nuclei labeled with Hoechst–JF549. The merged pseudo-color image (left; AF488 in cyan, JF549 in magenta) was extracted from the multispectral image. Scale bar: 10 µm. The phasor plot (middle) exhibits a largely bimodal cluster in which the two lobes sit near the two PCs. The mixing image (right) is largely uniformly cold, with only a region near the edge of the nucleus displaying any mixing. This small region reflects areas where the mitochondria extend over (or under) the nucleus but are still captured in the optical section. In the phasor plots, dashed gray lines indicate the bounds of the mixing band. The overlaid histograms display the S coordinate of the underling phasor cluster defined within the mixing band. (D) PCC, Manders' coefficients (M 1 and M 2 ) and the PMC (PMC 1 and PMC 2 ) for each example in A–C (from top to bottom). PCC and M 1,2 were calculated after applying an intensity threshold determined by the Otsu method (see Fig. S4 ). Importantly, PMC 1,2 capture the known association moving from high (top, A) to low (bottom, C) mixing. Individual N =3 points correspond to individual samples; line marker indicates the mean value.

Article Snippet: Rabbit IgG anti-TOMM20 (eLabScience, E-AB-81488) was diluted to 300 ng/ml in blocking buffer, and slides were incubated at 4°C in a humid, dark chamber overnight.

Techniques: Labeling, Marker

Journal: The FASEB Journal

Article Title: Increased mitochondrial fragmentation in polycystic kidney disease acts as a modifier of disease progression

doi: 10.1096/fj.201901739rr

Figure Lengend Snippet: FIGURE 4 Expression of mitochondrial fusion and fission proteins is altered in Ksp-Cre;Pkd1flox/− kidneys. A, Schematic representation of the molecular regulators of mitochondrial fusion and fission: pro-fusion proteins MFN1 on the OMM and OPA1 on the IMM, pro-fission protein DRP1, which is recruited from the cytosol to the mitochondria. B and C, Representative western blots for DRP1, MFN1, and OPA1 on total protein extracts from kidneys of Ksp-Cre;Pkd1flox/− mice (KO) and controls (Ctrl) at P4, P8, and P10. VINC and VDAC1 are used as loading controls for total and mitochondrial proteins, respectively. D-F, Densitometric analyses of western blots in B and C. Data represent mean ± SD of three independent experiments, t test, *P < .05, ***P < .001. G, Immunofluorescence on kidney sections with TOM20 as mitochondrial marker. DAPI and DBA are used to identify the nucleus and the distal tubules, respectively. Scale bar, 10 μm

Article Snippet: For immunofluorescence staining Lotus Tetragonolobus Lectin (LTL) (#FL-1321) and Dolichos Biflorus Agglutinin (DBA) (#RL-1032) are from Vector Laboratories (San Diego, CA, US) and used at 1:100; TOMM20 (#NBP1-81556) from Novus Biologicals (Centennial, CO, US) used at 1:60; DAPI (#SC-3598) from Santa Cruz Biotechnology (Dallas, TX, US), at 1:10 000.

Techniques: Expressing, Western Blot, Immunofluorescence, Marker