Journal: FEBS Open Bio

Article Title: Differential heme release from various hemoglobin redox states and the upregulation of cellular heme oxygenase‐1

doi: 10.1002/2211-5463.12103

Figure Lengend Snippet: Hemoglobin‐mediated induction of heme oxygenase‐1 ( HO ‐1) expressions in mouse E10 cells. E10 cells were exposed to either ferrous, ferric, or ferryl Hb at equimolar concentration (100 µ m ) for 12 h for immunocytochemistry experiment (A) or 24 h for western blotting experiment (B). Laser confocal images showing expression of heme oxygenase‐1 (green, Alexa‐488) in E10 cells (A). E10 cells were also counterstained with Phalloidin Alexa‐647 (red) to show the cellular morphology. Nuclear staining was done with DAPI (blue). Cell lysates were immunoblotted and analyzed for HO ‐1 expression (B, upper panel). Equal loading was confirmed by reprobing the blots against β‐actin (B, lower panel). The ratio of average ( n = 3) band intensity of HO ‐1 with corresponding β‐actin was plotted as relative intensity (C), * P < 0.05 vs. control; # P < 0.05 vs. HbFe 3+ , ‘ t ’ test unpaired.

Article Snippet: Cells were visualized under a Zeiss LSM710 meta confocal microscope (Zeiss, Thornwood, NY, USA) after mounting with Fluoroshield mounting medium with DAPI (Abcam).

Techniques: Concentration Assay, Immunocytochemistry, Western Blot, Expressing, Staining, Control

Journal: Molecular Neurodegeneration

Article Title: Tau oligomers impair memory and induce synaptic and mitochondrial dysfunction in wild-type mice

doi: 10.1186/1750-1326-6-39

Figure Lengend Snippet: Decrease in synaptophysin in mice injected with tau oligomers . Brain sections were stained with antibody to the presynaptic marker synaptophysin (green) nuclear staining with DAPI (blue). All images were taken from the CA1 region. Hemispheres injected with tau oligomers exhibited a decreased in synaptophysin (A). No decrease in the signal was observed in the hemispheres injected with fibrils (B), monomers (C), and PBS (D). (Scale bars = 10 μm.)

Article Snippet: The next day the sections were washed in PBS three times for 10 min and then incubated with goat anti-mouse IgG alexa488 (1:700; Invitrogen, A11004) for 1 h. Later, we washed the sections three times for 10 min in PBS, and finally we applied VECTASHIELD mounting medium with DAPI staining (Vector Lab., H-1000) and the slide was coverslipped and sealed with clear nail polish.

Techniques: Injection, Staining, Marker

Journal: Molecular Neurodegeneration

Article Title: Tau oligomers impair memory and induce synaptic and mitochondrial dysfunction in wild-type mice

doi: 10.1186/1750-1326-6-39

Figure Lengend Snippet: Caspase-9 activation in hemisphere injected with tau oligomers . Immunofluorescence using an antibody for pro-caspase-9 was performed. (A and B) Caspase-9 activation (green fluorescence) was observed in hemispheres injected with tau oligomers (A) or fibrils (B). However, the levels of activation were significantly higher in the hemispheres injected with oligomers compared with fibrils, which is not significant. (C and D). No activation of caspase-9 was observed in the hemispheres injected with tau monomers (C) or PBS (D). Nucleus was stained with DAPI (blue). (Scale bars = 10 μm.).

Article Snippet: The next day the sections were washed in PBS three times for 10 min and then incubated with goat anti-mouse IgG alexa488 (1:700; Invitrogen, A11004) for 1 h. Later, we washed the sections three times for 10 min in PBS, and finally we applied VECTASHIELD mounting medium with DAPI staining (Vector Lab., H-1000) and the slide was coverslipped and sealed with clear nail polish.

Techniques: Activation Assay, Injection, Immunofluorescence, Fluorescence, Staining

Journal: Oncology Letters

Article Title: Evaluation of a novel approach to circulating tumor cell isolation for cancer gene panel analysis in patients with breast cancer

doi: 10.3892/ol.2017.5807

Figure Lengend Snippet: Representative images of immunofluorescent staining of CTCs from patients with breast cancer. DAPI, 4′,6-diamidino-2-phenylindole; EpCAM, epithelial cell adhesion molecule; CD, cluster of differentiation; CTC, circulating tumor cell; WBC, white blood cell.

Article Snippet: The slides were mounted using FluoroshieldTM Mounting Medium with DAPI (ImmunoBioScience Corp., Mukilteo, WA, USA).

Techniques: Staining

Journal: bioRxiv

Article Title: Mitochondrial uncoupler and retinoic acid synergistically induce differentiation and inhibit proliferation in neuroblastoma

doi: 10.1101/2024.01.22.576741

Figure Lengend Snippet: (A) Left: morphological feature of neuroblastoma cells treated with indicated conditions for 96h (Scale bar: 50 μM). Right: Quantification of neurite outgrowth with NeuronJ. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, determined by the two-tailed Student’s t-test. (B) Immunofluorescence staining of β-tubulin III(Red) and DAPI (Blue) in cells treated with indicated condition for 96h. Scale bar: 25μm. (C) Cells in a 96-well plate were treated with a series of treatments, including RA, NEN, or their combination. After 72 hours, cell counts were normalized to controls. Drug combination effects were assessed using Chou-Talalay’s method and CompuSyn software, yielding combination index (CI) values. (D) Cells were pretreated with RA, NEN, or their combination. After 96 hours, cell viability was determined using trypan blue, and equal numbers of viable cells from each treatment group were replated in 96 well plate. Cell counts were performed at 48 and 96 hours using Cytation 5. Cells were kept atmospherically controlled in the imaging system under the same conditions as in the incubator.

Article Snippet: After two washes with PBS, cells were incubated with Alexa Fluor 594-conjugated anti-rabbit secondary antibody (Life Technologies) at RT for 1 h followed by staining with DAPI (Vector Laboratories, H-1800-2).

Techniques: Two Tailed Test, Immunofluorescence, Staining, Software, Imaging

Journal: International Journal of Nanomedicine

Article Title: Impact of Cerium Doping on the Osteogenic Properties of a 3D Biomimetic Piezoelectric Scaffold with Sustained Mg 2+ Release

doi: 10.2147/IJN.S514047

Figure Lengend Snippet: Biocompatibility evaluation of composite scaffolds. ( a ) Live/Dead cell staining and ( b ) phalloidin-DAPI staining of hBMSCs cultured with scaffold extracts at day 3 (nuclei – blue, cytoskeleton – red). ( c ) the proliferation of hBMSCs cultured with different scaffolds using CCK-8 assay.

Article Snippet: Cell adhesion and morphology on the scaffolds were examined as previously reported by staining nuclei with DAPI (Antifade Mounting Medium, P0131, Beyotime Biotechnology, China) and F-actin with phalloidin (Actin-Tracker Red-594, Beyotime Biotechnology, China).

Techniques: Staining, Cell Culture, CCK-8 Assay

Journal: Particle and Fibre Toxicology

Article Title: Oxidative stress generated by polycyclic aromatic hydrocarbons from ambient particulate matter enhance vascular smooth muscle cell migration through MMP upregulation and actin reorganization

doi: 10.1186/s12989-022-00472-z

Figure Lengend Snippet: Effect of organic matters from extracted ambient PM on VSMC migration. a Effects of seasonal organic matters from ambient PM on VSMC migration were measured using a wound healing assay (upper) and Boyden chamber assay (lower). The images from the wound healing assay are representative of five independent experiments, taken at the time of scratching and 48 h after scratching. The black line represents the initial boundaries following the scratch at time 0 and the white dotted line represents the migrating cell front after 48 h. The wound healing area was measured using ImageJ software. Images from the Boyden chamber assay shows the number of migrated cells. The images from the transwell assay are representative of five independent experiments, taken 12 h after seeding. b The effects of ambient PM on VSMC proliferation were evaluated using the BrdU incorporation assay. Serum-starved VSMCs were treated with negative control (0.1% DMSO), SPM, WPM or PDGF-BB (20 ng/mL) as a positive control for 24 h. c ROS generation in PM-treated VSMCs was evaluated using H 2 DCF-DA (green). VSMCs were pretreated with or without NAC (1 mM) for 1 h, then treated with negative control (0.1% DMSO), SPM or WPM. Nuclei were stained with DAPI (blue). Fluorescence intensity was quantified by SIBIA software. d Effects of the antioxidant, NAC, on organic matters from ambient PM-induced VSMC migration were measured using a wound healing assay (upper) and Boyden chamber assay (lower). All values are represented as mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001 versus control; # P < 0.05, ## P < 0.01, and ### P < 0.001 WPM versus SPM; $ P < 0.05, $$ P < 0.01, and $$$ P < 0.001 PM with NAC versus PM alone; NS, no significance

Article Snippet: Then, the cells were gently washed under the cover slip three times with PBS and mounted in fluoroshieldTM mounting medium with DAPI (Cell Signaling, Beverly, MA, US).

Techniques: Migration, Wound Healing Assay, Boyden Chamber Assay, Software, Transwell Assay, BrdU Incorporation Assay, Negative Control, Positive Control, Staining, Fluorescence, Control

Journal: Particle and Fibre Toxicology

Article Title: Oxidative stress generated by polycyclic aromatic hydrocarbons from ambient particulate matter enhance vascular smooth muscle cell migration through MMP upregulation and actin reorganization

doi: 10.1186/s12989-022-00472-z

Figure Lengend Snippet: Effect of PAHs on ROS generation and VSMCs migration. a VSMCs were treated with control (DMSO), ANT, AQ, BaA, or BAQ at the indicated concentration and analyzed for ROS generation. Representative fluorescent images of VSMCs loaded with H 2 DCF-DA (green) with nuclei stained with DAPI (blue) are shown. b Effects of ambient PM on VSMC migration were measured using a wound healing assay. The images from the wound healing assay are representative of five independent experiments, taken at the time of scratching and 48 h after scratching. The black line represents the initial boundaries following the scratch at time 0 and the white dotted line represents the migrating cell front after 48 h. The wound healing area was measured using ImageJ software. c Boyden chamber assay showing the number of migrated cells. The images from the transwell assay are representative of five independent experiments, taken 12 h after seeding. d The cytotoxic effects of PAHs on VSMCs were evaluated using the MTT assay. Serum-starved VSMCs were treated with each form of PAHs for 24 h. All values are represented as mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001 versus control; # P < 0.05 and ## P < 0.01 oxy-PAHs versus PAHs; NS, no significance

Article Snippet: Then, the cells were gently washed under the cover slip three times with PBS and mounted in fluoroshieldTM mounting medium with DAPI (Cell Signaling, Beverly, MA, US).

Techniques: Migration, Control, Concentration Assay, Staining, Wound Healing Assay, Software, Boyden Chamber Assay, Transwell Assay, MTT Assay