Journal: Advanced Science

Article Title: Delivering Antisense Oligonucleotides across the Blood‐Brain Barrier by Tumor Cell‐Derived Small Apoptotic Bodies

doi: 10.1002/advs.202004929

Figure Lengend Snippet: sCABs are transcytosed by BMECs to cross the BBB. a) TEM images showing a phagosome containing a sCAB in the BMEC of a mouse 10 min after an i.v. sCABs injection. The right panel shows a magnified view of the phagophore selected by the red square and arrow. Scale bar: 500 nm. b) TEM images showing the structure of TJs after sCABs passed through microvessels 15 min after an i.v. sCABs injection. The TJs in the red squares are shown at high magnifications on the right. The red arrow depicts sCABs that passed through BMECs. Scale bar: 500 nm. c) Representative fluorescence images showing the intact structure of TJs after sCABs treatment containing Cy5‐ASO. The right panels show magnified views of the area selected by the white square. Scale bar: 25 µm. d) Representative fluorescence microscopy images showing sCABs containing Cy5‐ASO phagocytized by b. End3 cells in the BBB model. Scale bar: 25 µm. e) Representative fluorescence images showing Cy5‐ASO delivered by sCABs but not the naked one was phagocytized by microglial cells in the BBB model 24 h after the incubation. The morphology of microglial cells as imaged with differentia linterference contrast (DIC). Scale bar: 25 µm. f) Fluorescence images showing GFP‐labeled sCABs containing Cy5‐ASO phagocytized by microglia 24 h after the incubation. The right panels show magnifications of the area selected by the white square. Scale bar: 10 µm. g–k) Representative fluorescence images showing the colocalization of sCABs containing Cy5‐ASO with special protein markers ((g) labeled with clathrin, (h) with caveolin‐1, (i) with EEA‐1, (h) with Rab11 involved in endocytosis, and (k) with Snap23). The colocalization of clathrin and caveolin‐1 was examined at 5 min after sCABs incubation, EEA‐1 and Rab11 at 10 min, and Snap23 at 20 min. The right panels show magnifications of the areas selected by the white square. Scale bar: 25 µm. Images are representative of three independent experiments.

Article Snippet: EEA‐1 (anti‐EEA‐1 antibody, BM4402, Boster, China), an intracellular marker of early endosomes, was tested at 10 min, as was Rab11 (anti‐Rab11B antibody, A04526‐1, Boster, China), a marker of recycling endosomes.

Techniques: Injection, Fluorescence, Microscopy, Incubation, Labeling

Journal: eLife

Article Title: The glycosphingolipid MacCer promotes synaptic bouton formation in Drosophila by interacting with Wnt

doi: 10.7554/eLife.38183

Figure Lengend Snippet: ( A–D ) Confocal images of single slice of NMJ4 boutons triple-labeled with anti-MacCer (red), anti-HRP (blue) and anti-Wg (green; A–B ) or anti-Rab11 (green; C–D ) in wild type and egh 62d18 mutants. MacCer puncta showed substantial colocalization (arrowheads) with Wg and Rab11 in boutons. Arrows indicate Rab11-positive puncta without MacCer staining in the egh 62d18 mutants. Images were processed by deconvolution. Scale bar: 2 μm. ( E ) Pearson’s coefficients of colocalization between MacCer and indicated proteins. n = 51 and 44 boutons (from ten wild-type larvae each) for colocalization of MacCer with Wg and Rab11, respectively. ( F–H ) Representative images of NMJ4 co-labeled with anti-Wg (green) and anti-HRP (magenta) in wild type ( F ), egh 62d18 mutants ( G ) and UAS-Egh/+; nSyb-Gal4/+ ( H ). Scale bar: 5 μm. ( I ) Quantification of intensities of endogenous Wg normalized to HRP intensities in different genotypes. n ≥ 15 larvae; ns, no significance; **p<0.01 and ***p<0.001 by student’s t test; error bars: s.e.m. ( J–L ) Representative images of NMJ6/7 labeled with anti-Futsch (green) and anti-HRP (magenta) in wild type ( J ), egh 62d18 mutants ( K ) and egh 62d18 /+; wg 1 / +transheterozygotes ( L ). Insets show higher magnification images of Futsch staining of a single bouton (asterisk). Arrows indicate boutons displaying unbundled Futsch; arrowheads denote boutons with Futsch loops. Scale bar: 10 μm. ( M ) Quantifications of percentage of Futsch loops and unbundled Futsch staining in different genotypes. The genotype of egh rescue was egh 62d18 ; UAS-Egh/+; nSyb-Gal4/+ ; the genotype of wg was wg 1 / wg CX4 . n ≥ 12 larvae; ns, no significance, *p<0.05; **p<0.01 by one-way ANOVA with Tukey post hoc tests, error bars: s.e.m. 10.7554/eLife.38183.022 Figure 6—source data 1. Numerical data for the statistical graphs.

Article Snippet: For GluRIIA staining, specimens were fixed in cold methanol for 5 min. We used the following antibodies: mouse anti-cysteine string protein (CSP) (1:300; 6D6 from DSHB), mouse anti-Wg (1:10; 4D4 from DSHB), mouse anti-Futsch (1:50; 22C10 from DSHB), mouse anti-Rab11 (1:50; BD Biosciences), mouse anti-DLG (1:500; 4F3 from DSHB), mouse anti-GluRIIA (1:200; 8B4D2 from DSHB), mouse anti-Fas II (1:50; 1D4 from DSHB), mouse anti-Syx1A (1:20; 8C3 from DSHB), mouse anti-dFz2 (1:5; 12A7 from DSHB), mouse anti-HA (1:1000; MBL International), mouse anti-Myc (1:300; CWBIO, MBL International), rat anti-GFP (1:200; MBL International), and fluorescence-conjugated anti-horseradish peroxidase (HRP) (1:200; Jackson ImmunoResearch).

Techniques: Labeling, Staining

Journal: eLife

Article Title: The glycosphingolipid MacCer promotes synaptic bouton formation in Drosophila by interacting with Wnt

doi: 10.7554/eLife.38183

Figure Lengend Snippet:

Article Snippet: For GluRIIA staining, specimens were fixed in cold methanol for 5 min. We used the following antibodies: mouse anti-cysteine string protein (CSP) (1:300; 6D6 from DSHB), mouse anti-Wg (1:10; 4D4 from DSHB), mouse anti-Futsch (1:50; 22C10 from DSHB), mouse anti-Rab11 (1:50; BD Biosciences), mouse anti-DLG (1:500; 4F3 from DSHB), mouse anti-GluRIIA (1:200; 8B4D2 from DSHB), mouse anti-Fas II (1:50; 1D4 from DSHB), mouse anti-Syx1A (1:20; 8C3 from DSHB), mouse anti-dFz2 (1:5; 12A7 from DSHB), mouse anti-HA (1:1000; MBL International), mouse anti-Myc (1:300; CWBIO, MBL International), rat anti-GFP (1:200; MBL International), and fluorescence-conjugated anti-horseradish peroxidase (HRP) (1:200; Jackson ImmunoResearch).

Techniques: Mutagenesis, Fluorescence, Recombinant, High Performance Thin Layer Chromatography, Software