Journal: Journal of Mammary Gland Biology and Neoplasia

Article Title: Methods for investigating STAT3 regulation of lysosomal function in mammary epithelial cells

doi: 10.1007/s10911-024-09563-3

Figure Lengend Snippet: Schematic overview of the lysosomal leakiness assay. The permeability of isolated lysosomes can be assessed by western blotting for cathepsin B and L antibodies to visualise the degree of leakiness of cathepsin hydrolases into the supernatant fraction under different treatment conditions (i.e. vehicle and OSM stimulated conditions in EpH4 cells). Immunoblotting for LAMP2, a lysosomal membrane protein, can be used to standardise the amount of lysosomal material in a pellet during the assay. Alternatively, cathepsin activity levels can be assessed in supernatant and pellet fractions using enzyme activity assays

Article Snippet: Primary and secondary antibodies as required e.g. rat anti-LAMP2 antibody (GL2A7, ab13524, Abcam, 1:200), rat anti-LAMP1 (ID4B, Developmental Studies Hybridoma Bank, 1:200).

Techniques: Permeability, Isolation, Western Blot, Membrane, Activity Assay

Journal: Journal of Mammary Gland Biology and Neoplasia

Article Title: Methods for investigating STAT3 regulation of lysosomal function in mammary epithelial cells

doi: 10.1007/s10911-024-09563-3

Figure Lengend Snippet: STAT3-mediated upregulation of the vesicular compartment in EpH4 cells. A Fluorescence microscopy of LAMP1 and LAMP2 immunostaining in EpH4 cells treated with OSM for 72 h show upregulation of the lysosomal compartment with STAT3 activation. Scale bars: 20 μm. B Fluorescence microscopy of LysoTracker® staining in EpH4 cells treated with OSM for 72 h show upregulation of the acidic compartment with STAT3 activation. Scale bars: 20 μm. C Transmission electron microscopy images of EpH4 cells showing an increased number of degradative vesicles after 72 h of OSM stimulation. Scale bars: 500 nm

Article Snippet: Primary and secondary antibodies as required e.g. rat anti-LAMP2 antibody (GL2A7, ab13524, Abcam, 1:200), rat anti-LAMP1 (ID4B, Developmental Studies Hybridoma Bank, 1:200).

Techniques: Fluorescence, Microscopy, Immunostaining, Activation Assay, Staining, Transmission Assay, Electron Microscopy

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: