Structured Review

Carl Zeiss lsm 510 confocal microscope
Heterozygosity for Cldn14 increases tumour blood vessel leakage and decreases intratumoural hypoxia. Wild-type, Cldn14-heterozygous and Cldn14-null mice were injected subcutaneously in the flank with 0.5×10 6 B16F10 melanoma or Lewis Lung Carcinoma (LLC) cells. ( A ) At 10 days post inoculation, PE-conjugated anti-PECAM antibody and Hoechst dye were injected via the tail vein prior to sacrifice. Midline sections (100 µm) of snap-frozen tumours were fixed, mounted and imaged using a Zeiss <t>LSM</t> 510 confocal microscope. The extent of Hoechst leakage was measured in z-stacks using ImageJ. Bars show mean Hoechst leakage relative to PECAM signal ± SEM. Blood vessel leakage is increased significantly in Cldn14-het mice when compared with WT and Cldn14-null mice. ( B ) Representative images of Hoechst (blue) and PECAM (red) detection. ( C ) Tumour-bearing mice from each genotype were injected with pimonidazole prior to sacrifice to measure hypoxic areas within the tumour. 8 µm tumour cryosections were then double stained with anti-pimonidazole antibody (green) to highlight hypoxic areas and anti-PECAM antibody to identify blood vessels. The hypoxic index was quantified relative to PECAM staining using image J software. Bars represent mean relative hypoxic index ± SEM. ( D ) Representative images of pimonidazole detection and PECAM-positive blood vessels in tumour sections. Arrows , blood vessels; Asterisks , pimonidazole-positive staining. Scale bars: A 50 µm; D 200 µm. N = 4 tumours per genotype. NSD: not statistically different, * P
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Images

1) Product Images from "Stromal Claudin14-Heterozygosity, but Not Deletion, Increases Tumour Blood Leakage without Affecting Tumour Growth"

Article Title: Stromal Claudin14-Heterozygosity, but Not Deletion, Increases Tumour Blood Leakage without Affecting Tumour Growth

Journal: PLoS ONE

doi: 10.1371/journal.pone.0062516

Heterozygosity for Cldn14 increases tumour blood vessel leakage and decreases intratumoural hypoxia. Wild-type, Cldn14-heterozygous and Cldn14-null mice were injected subcutaneously in the flank with 0.5×10 6 B16F10 melanoma or Lewis Lung Carcinoma (LLC) cells. ( A ) At 10 days post inoculation, PE-conjugated anti-PECAM antibody and Hoechst dye were injected via the tail vein prior to sacrifice. Midline sections (100 µm) of snap-frozen tumours were fixed, mounted and imaged using a Zeiss LSM 510 confocal microscope. The extent of Hoechst leakage was measured in z-stacks using ImageJ. Bars show mean Hoechst leakage relative to PECAM signal ± SEM. Blood vessel leakage is increased significantly in Cldn14-het mice when compared with WT and Cldn14-null mice. ( B ) Representative images of Hoechst (blue) and PECAM (red) detection. ( C ) Tumour-bearing mice from each genotype were injected with pimonidazole prior to sacrifice to measure hypoxic areas within the tumour. 8 µm tumour cryosections were then double stained with anti-pimonidazole antibody (green) to highlight hypoxic areas and anti-PECAM antibody to identify blood vessels. The hypoxic index was quantified relative to PECAM staining using image J software. Bars represent mean relative hypoxic index ± SEM. ( D ) Representative images of pimonidazole detection and PECAM-positive blood vessels in tumour sections. Arrows , blood vessels; Asterisks , pimonidazole-positive staining. Scale bars: A 50 µm; D 200 µm. N = 4 tumours per genotype. NSD: not statistically different, * P
Figure Legend Snippet: Heterozygosity for Cldn14 increases tumour blood vessel leakage and decreases intratumoural hypoxia. Wild-type, Cldn14-heterozygous and Cldn14-null mice were injected subcutaneously in the flank with 0.5×10 6 B16F10 melanoma or Lewis Lung Carcinoma (LLC) cells. ( A ) At 10 days post inoculation, PE-conjugated anti-PECAM antibody and Hoechst dye were injected via the tail vein prior to sacrifice. Midline sections (100 µm) of snap-frozen tumours were fixed, mounted and imaged using a Zeiss LSM 510 confocal microscope. The extent of Hoechst leakage was measured in z-stacks using ImageJ. Bars show mean Hoechst leakage relative to PECAM signal ± SEM. Blood vessel leakage is increased significantly in Cldn14-het mice when compared with WT and Cldn14-null mice. ( B ) Representative images of Hoechst (blue) and PECAM (red) detection. ( C ) Tumour-bearing mice from each genotype were injected with pimonidazole prior to sacrifice to measure hypoxic areas within the tumour. 8 µm tumour cryosections were then double stained with anti-pimonidazole antibody (green) to highlight hypoxic areas and anti-PECAM antibody to identify blood vessels. The hypoxic index was quantified relative to PECAM staining using image J software. Bars represent mean relative hypoxic index ± SEM. ( D ) Representative images of pimonidazole detection and PECAM-positive blood vessels in tumour sections. Arrows , blood vessels; Asterisks , pimonidazole-positive staining. Scale bars: A 50 µm; D 200 µm. N = 4 tumours per genotype. NSD: not statistically different, * P

Techniques Used: Mouse Assay, Injection, Microscopy, Staining, Software

2) Product Images from "Two Hydroxyproline Galactosyltransferases, GALT5 and GALT2, Function in Arabinogalactan-Protein Glycosylation, Growth and Development in Arabidopsis"

Article Title: Two Hydroxyproline Galactosyltransferases, GALT5 and GALT2, Function in Arabinogalactan-Protein Glycosylation, Growth and Development in Arabidopsis

Journal: PLoS ONE

doi: 10.1371/journal.pone.0125624

Staining of seed coat mucilage for cellulose and pectin in wild type, galt , sos5 , and fei mutant seeds. Seeds of the indicated genotypes were prehydrated with water and stained with Calcofluor white and ruthenium red to visualize cellulose and pectin with a Zeiss LSM 510 META laser scanning confocal microscope.
Figure Legend Snippet: Staining of seed coat mucilage for cellulose and pectin in wild type, galt , sos5 , and fei mutant seeds. Seeds of the indicated genotypes were prehydrated with water and stained with Calcofluor white and ruthenium red to visualize cellulose and pectin with a Zeiss LSM 510 META laser scanning confocal microscope.

Techniques Used: Staining, Mutagenesis, Microscopy

3) Product Images from "Golgi Complex Reorganization during Muscle Differentiation: Visualization in Living Cells and Mechanism"

Article Title: Golgi Complex Reorganization during Muscle Differentiation: Visualization in Living Cells and Mechanism

Journal: Molecular Biology of the Cell

doi:

FRAP of mannosidase-GFP shows breakdown of continuity during differentiation. C2 cells that express man-GFP were pretreated with 25 μg/ml cycloheximide to inhibit protein synthesis and examined in the LSM 510 confocal microscope at 37°C. Each series of images shows a view before (pre), immediately after (5 s), and at different times after photobleaching. (A) Fast recovery of a 2-μm-wide band is observed in myoblasts (arrowhead). The fluorescence intensity of the bleached area was measured with NIH Image, and plotted and fitted in Kaleidagraph (see graph) to determine the diffusion coefficient (see MATERIALS AND METHODS and text). (B) In differentiated myotubes, fragments of individual elements (arrows) or whole elements (arrowheads) were photobleached. Recovery was fast for the fragments connected to a GFP source but absent for the whole elements. In C, the entire GC of a myoblast was photobleached and in D all elements of an extended area of myotubes. A slower and less complete recovery was observed, as plotted in the graph. Bars, 10 μm.
Figure Legend Snippet: FRAP of mannosidase-GFP shows breakdown of continuity during differentiation. C2 cells that express man-GFP were pretreated with 25 μg/ml cycloheximide to inhibit protein synthesis and examined in the LSM 510 confocal microscope at 37°C. Each series of images shows a view before (pre), immediately after (5 s), and at different times after photobleaching. (A) Fast recovery of a 2-μm-wide band is observed in myoblasts (arrowhead). The fluorescence intensity of the bleached area was measured with NIH Image, and plotted and fitted in Kaleidagraph (see graph) to determine the diffusion coefficient (see MATERIALS AND METHODS and text). (B) In differentiated myotubes, fragments of individual elements (arrows) or whole elements (arrowheads) were photobleached. Recovery was fast for the fragments connected to a GFP source but absent for the whole elements. In C, the entire GC of a myoblast was photobleached and in D all elements of an extended area of myotubes. A slower and less complete recovery was observed, as plotted in the graph. Bars, 10 μm.

Techniques Used: Microscopy, Fluorescence, Diffusion-based Assay

4) Product Images from "Inhibition of EZH2 methyltransferase decreases immunoediting of mesothelioma cells by autologous macrophages through a PD-1–dependent mechanism"

Article Title: Inhibition of EZH2 methyltransferase decreases immunoediting of mesothelioma cells by autologous macrophages through a PD-1–dependent mechanism

Journal: JCI Insight

doi: 10.1172/jci.insight.128474

Direct cytotoxicity of RAW264.7 macrophages upon cell-to-cell contact with syngeneic mesothelioma AB1 cells. ( A ) Experimental design. RAW264.7 macrophages were treated with L-NMMA or apocynin for 24 hours and then further cultivated in the presence or absence of LPS for 24 hours. After 3 washes in PBS, RAW264.7 macrophages were cocultivated with AB1 cells at a 10:1 ratio for 48 hours. ( B ) AB1 cells and CFSE-labeled RAW264.7 macrophages were monitored by time-lapse microscopy using an LSM 510 (Zeiss) equipped with an environmental chamber maintained at 37°C in a humidified 5% CO 2 atmosphere. ( C ) Cells were fixed, permeabilized, and stained for F4/80 (shown in green) and nitrosylated tyrosine (N-Tyr; shown in blue). Images were acquired using a Zeiss LSM 510 confocal microscope equipped with a ×63-1.4 oil immersion objective. ( D ) Apoptotic rates of AB1 cells were determined by flow cytometry after staining with the annexin V-FITC kit (Becton Dickinson). Each bar represents the mean ± SD from 8 independent experiments performed in triplicate. ( E ) AB1 cells were transduced by lentivectors encoding PGAM5 shRNAs (#2 and #5) or a scramble control. The levels of PGAM5 transcripts were measured by reverse transcription quantitative PCR. ( F ) RAW264.7-induced apoptosis of shRNA-transduced AB1 cells was measured as described in D . Each bar represents the mean ± SD from 6 independent experiments. Statistical significance was evaluated using 1-way ANOVA followed by Tukey’s multiple-comparisons test. * P
Figure Legend Snippet: Direct cytotoxicity of RAW264.7 macrophages upon cell-to-cell contact with syngeneic mesothelioma AB1 cells. ( A ) Experimental design. RAW264.7 macrophages were treated with L-NMMA or apocynin for 24 hours and then further cultivated in the presence or absence of LPS for 24 hours. After 3 washes in PBS, RAW264.7 macrophages were cocultivated with AB1 cells at a 10:1 ratio for 48 hours. ( B ) AB1 cells and CFSE-labeled RAW264.7 macrophages were monitored by time-lapse microscopy using an LSM 510 (Zeiss) equipped with an environmental chamber maintained at 37°C in a humidified 5% CO 2 atmosphere. ( C ) Cells were fixed, permeabilized, and stained for F4/80 (shown in green) and nitrosylated tyrosine (N-Tyr; shown in blue). Images were acquired using a Zeiss LSM 510 confocal microscope equipped with a ×63-1.4 oil immersion objective. ( D ) Apoptotic rates of AB1 cells were determined by flow cytometry after staining with the annexin V-FITC kit (Becton Dickinson). Each bar represents the mean ± SD from 8 independent experiments performed in triplicate. ( E ) AB1 cells were transduced by lentivectors encoding PGAM5 shRNAs (#2 and #5) or a scramble control. The levels of PGAM5 transcripts were measured by reverse transcription quantitative PCR. ( F ) RAW264.7-induced apoptosis of shRNA-transduced AB1 cells was measured as described in D . Each bar represents the mean ± SD from 6 independent experiments. Statistical significance was evaluated using 1-way ANOVA followed by Tukey’s multiple-comparisons test. * P

Techniques Used: Labeling, Time-lapse Microscopy, Staining, Microscopy, Flow Cytometry, Cytometry, Real-time Polymerase Chain Reaction, shRNA

5) Product Images from "Mutations in CHMP2B in Lower Motor Neuron Predominant Amyotrophic Lateral Sclerosis (ALS)"

Article Title: Mutations in CHMP2B in Lower Motor Neuron Predominant Amyotrophic Lateral Sclerosis (ALS)

Journal: PLoS ONE

doi: 10.1371/journal.pone.0009872

Overexpression of mutant CHMP2B produces an aberrant phenotype in HEK-293 cells. Cells were transfected with vectors encoding recombinant protein c-Myc-CHMP2B with either the wild-type or I29V, T104N or Q206H mutant sequence, and stained with FITC-conjugated antibody to c-Myc. Transfection with wild-type CHMP2B (A) results in generalised cytoplasmic expression, whereas the mutant isoforms I29V (B), T104N (C) and Q206H (D) resulted in cytoplasmic vacuoles of varying size (indicated by arrowheads). Another striking observation was the presence within cells expression mutant CHMP2B of circular CHMP2B accumulations in the cytoplasm, termed halos (E). Cells were doubly stained with antibodies to c-Myc (F I), as well as CD63 (G J), and merged to show co-localisation (H K). CD63 co-localises with the small vacuoles found in cells transfected with WT CHMP2B (F–H). However, CD63 staining does not co-localise with large vacuoles in mutant expressing cells (cells transfected with T104N shown), but are found on the vacuole edge (I–K). Images were taken on a Zeiss LSM 510 confocal microscope, ×63 obj. Bar, 10µm.
Figure Legend Snippet: Overexpression of mutant CHMP2B produces an aberrant phenotype in HEK-293 cells. Cells were transfected with vectors encoding recombinant protein c-Myc-CHMP2B with either the wild-type or I29V, T104N or Q206H mutant sequence, and stained with FITC-conjugated antibody to c-Myc. Transfection with wild-type CHMP2B (A) results in generalised cytoplasmic expression, whereas the mutant isoforms I29V (B), T104N (C) and Q206H (D) resulted in cytoplasmic vacuoles of varying size (indicated by arrowheads). Another striking observation was the presence within cells expression mutant CHMP2B of circular CHMP2B accumulations in the cytoplasm, termed halos (E). Cells were doubly stained with antibodies to c-Myc (F I), as well as CD63 (G J), and merged to show co-localisation (H K). CD63 co-localises with the small vacuoles found in cells transfected with WT CHMP2B (F–H). However, CD63 staining does not co-localise with large vacuoles in mutant expressing cells (cells transfected with T104N shown), but are found on the vacuole edge (I–K). Images were taken on a Zeiss LSM 510 confocal microscope, ×63 obj. Bar, 10µm.

Techniques Used: Over Expression, Mutagenesis, Transfection, Recombinant, Sequencing, Staining, Expressing, Microscopy

6) Product Images from "Targeting endoplasmic reticulum protein transport: a novel strategy to kill malignant B cells and overcome fludarabine resistance in CLL"

Article Title: Targeting endoplasmic reticulum protein transport: a novel strategy to kill malignant B cells and overcome fludarabine resistance in CLL

Journal: Blood

doi: 10.1182/blood-2005-05-1923

Activation of multiple caspases by brefeldin A. (A) Caspase-2 is localized to the Golgi apparatus and nuclear membrane in primary CLL cells. Control and brefeldin A-treated (100 ng/mL, 24h) B-CLL cells were cytocentrifuged onto glass slides, fixed, and stained with a monoclonal human anti-caspase-2 antibody and markers for the nucleus (To-Pro-3) and Golgi apparatus (BODIPY-TR-Ceramide). Subcellular localization of caspase-2 was visualized by confocal microscopy using an LSM 510 confocal microscope with a Plan-Neofluar dry 40 ×/0.75 objective lens and a built-in camera (Carl Zeiss, Thornwood, NY). (B) BFA treatment leads to activation of caspases-2, -8, -9, and -3. Lysates were prepared from cells with or without 100 ng/mL BFA treatment for 24 hours. Western blotting was used to evaluate the indicated caspases in primary CLL cells and U266 cells. The antibody used for caspase-3 recognizes the cleaved (active) form only. Actin was used as a loading control. NR = nonrefractory; R = fludarabine refractory.
Figure Legend Snippet: Activation of multiple caspases by brefeldin A. (A) Caspase-2 is localized to the Golgi apparatus and nuclear membrane in primary CLL cells. Control and brefeldin A-treated (100 ng/mL, 24h) B-CLL cells were cytocentrifuged onto glass slides, fixed, and stained with a monoclonal human anti-caspase-2 antibody and markers for the nucleus (To-Pro-3) and Golgi apparatus (BODIPY-TR-Ceramide). Subcellular localization of caspase-2 was visualized by confocal microscopy using an LSM 510 confocal microscope with a Plan-Neofluar dry 40 ×/0.75 objective lens and a built-in camera (Carl Zeiss, Thornwood, NY). (B) BFA treatment leads to activation of caspases-2, -8, -9, and -3. Lysates were prepared from cells with or without 100 ng/mL BFA treatment for 24 hours. Western blotting was used to evaluate the indicated caspases in primary CLL cells and U266 cells. The antibody used for caspase-3 recognizes the cleaved (active) form only. Actin was used as a loading control. NR = nonrefractory; R = fludarabine refractory.

Techniques Used: Activation Assay, Staining, Confocal Microscopy, Microscopy, Western Blot

7) Product Images from "Hypoxia, Inflammation and Necrosis as Determinants of Glioblastoma Cancer Stem Cells Progression"

Article Title: Hypoxia, Inflammation and Necrosis as Determinants of Glioblastoma Cancer Stem Cells Progression

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms21082660

Hypoxia-induced RAGE expression is prevented by HIF-1α inhibition. ( A ) GSC #1 was grown on glass coverslips as indicated in Materials and Methods. Afterward, cells were kept under normoxia or hypoxia for 24 h in the presence or absence of 100 nM Digoxin and 5 µM Acriflavine and then processed for RAGE immunofluorescence staining as indicated in Materials and Methods. Images were taken with a Zeiss LSM 510 confocal microscope (Magnification 60×). ( B ) GSC #83 was grown on glass coverslips as indicated in Materials and Methods. Afterward, cells were kept under normoxia or hypoxia for 24 h in the presence or absence of 200 nM Digoxin and 10 µM Acriflavine and then processed for RAGE immunofluorescence staining as indicated in Materials and Methods. Images were taken with a Zeiss LSM 510 confocal microscope (Magnification 60×).
Figure Legend Snippet: Hypoxia-induced RAGE expression is prevented by HIF-1α inhibition. ( A ) GSC #1 was grown on glass coverslips as indicated in Materials and Methods. Afterward, cells were kept under normoxia or hypoxia for 24 h in the presence or absence of 100 nM Digoxin and 5 µM Acriflavine and then processed for RAGE immunofluorescence staining as indicated in Materials and Methods. Images were taken with a Zeiss LSM 510 confocal microscope (Magnification 60×). ( B ) GSC #83 was grown on glass coverslips as indicated in Materials and Methods. Afterward, cells were kept under normoxia or hypoxia for 24 h in the presence or absence of 200 nM Digoxin and 10 µM Acriflavine and then processed for RAGE immunofluorescence staining as indicated in Materials and Methods. Images were taken with a Zeiss LSM 510 confocal microscope (Magnification 60×).

Techniques Used: Expressing, Inhibition, Immunofluorescence, Staining, Microscopy

8) Product Images from "Quantum dot assisted tracking of the intracellular protein Cyclin E in Xenopus laevis embryos"

Article Title: Quantum dot assisted tracking of the intracellular protein Cyclin E in Xenopus laevis embryos

Journal: Journal of Nanobiotechnology

doi: 10.1186/s12951-015-0092-6

Localization of (QD 564 )-His 6 Cyclin E in live pre-MBT (4 hpf, 64-cell embryo, a-c ) and MBT (6 hpf, 2048-cell embryo, d-f ) Xenopus laevis embryos. One cell of embryos at the 2-cell stage was microinjected with (QD 564 )-His 6 Cyclin E and visualized using confocal microscopy. (a, d) fluorescence channel; (b, e) light channel; (c, f) merged fluorescence and light channels. Nuclei are marked with white arrowheads in panels b, f. Embryos were viewed with a 10X objective on a Zeiss LSM 510 confocal microscope equipped with a META detector, and analyzed using LSM510 Image Acquisition software. Scale bars are 100 μM. At least 20 embryos were injected and viewed in at least 3 separate experiments.
Figure Legend Snippet: Localization of (QD 564 )-His 6 Cyclin E in live pre-MBT (4 hpf, 64-cell embryo, a-c ) and MBT (6 hpf, 2048-cell embryo, d-f ) Xenopus laevis embryos. One cell of embryos at the 2-cell stage was microinjected with (QD 564 )-His 6 Cyclin E and visualized using confocal microscopy. (a, d) fluorescence channel; (b, e) light channel; (c, f) merged fluorescence and light channels. Nuclei are marked with white arrowheads in panels b, f. Embryos were viewed with a 10X objective on a Zeiss LSM 510 confocal microscope equipped with a META detector, and analyzed using LSM510 Image Acquisition software. Scale bars are 100 μM. At least 20 embryos were injected and viewed in at least 3 separate experiments.

Techniques Used: Confocal Microscopy, Fluorescence, Microscopy, Software, Injection

Localization of exogenous Cyclin E in pre-MBT and MBT Xenopus laevis embryos. One cell of 2-cell embryo was microinjected with in vitro transcribed Myc 6− GFP-Cyclin E RNA, collected at indicated time points, and the translated protein detected in fixed and stained embryos. For immunofluorescence analysis of Cyclin E localization, embryos were collected at 4 hpf, pre-MBT (a-c) or at 6 hpf, MBT (d-f). (a, d) Embryos were fixed and stained with an antibody against the Myc 6 tag (αMyc) followed by an Alexa488 conjugated secondary antibody. (b, e) Embryos were counterstained with DAPI to visualize the nuclei. (c, f) . Merged image of the Alexa488 and DAPI. White arrowheads in d-f indicate nuclei. Embryos were viewed with a 10X objective on a Zeiss LSM 510 confocal microscope equipped with a META detector, and analyzed using LSM510 Image Acquisition software. Scale bars are 100 μM. At least 20 embryos were injected in at least 3 separate experiments, with at least 5 embryos fixed per timepoint for analysis.
Figure Legend Snippet: Localization of exogenous Cyclin E in pre-MBT and MBT Xenopus laevis embryos. One cell of 2-cell embryo was microinjected with in vitro transcribed Myc 6− GFP-Cyclin E RNA, collected at indicated time points, and the translated protein detected in fixed and stained embryos. For immunofluorescence analysis of Cyclin E localization, embryos were collected at 4 hpf, pre-MBT (a-c) or at 6 hpf, MBT (d-f). (a, d) Embryos were fixed and stained with an antibody against the Myc 6 tag (αMyc) followed by an Alexa488 conjugated secondary antibody. (b, e) Embryos were counterstained with DAPI to visualize the nuclei. (c, f) . Merged image of the Alexa488 and DAPI. White arrowheads in d-f indicate nuclei. Embryos were viewed with a 10X objective on a Zeiss LSM 510 confocal microscope equipped with a META detector, and analyzed using LSM510 Image Acquisition software. Scale bars are 100 μM. At least 20 embryos were injected in at least 3 separate experiments, with at least 5 embryos fixed per timepoint for analysis.

Techniques Used: In Vitro, Staining, Immunofluorescence, Microscopy, Software, Injection

Cyclin E accumulates in the nucleus of live Xenopus laevis embryos at the MBT (6 hpf). One cell of the 2-cell embryo was microinjected with in vitro transcribed Myc 6 -GFP-Cyclin E RNA and the translated protein visualized in live embryos using confocal microscopy in real time. (a, b) Fluorescence channel, Z stack images #5 and #8 from the top, respectively. Nuclei are marked with white arrowheads. (c) Light field image. A 3D image is shown. Scale bars are 100 μM. Embryos were viewed with a 10X objective on a Zeiss LSM 510 confocal microscope equipped with a META detector, and analyzed using LSM510 Image Acquisition software. At least 20 embryos were injected in at least 3 separate experiments.
Figure Legend Snippet: Cyclin E accumulates in the nucleus of live Xenopus laevis embryos at the MBT (6 hpf). One cell of the 2-cell embryo was microinjected with in vitro transcribed Myc 6 -GFP-Cyclin E RNA and the translated protein visualized in live embryos using confocal microscopy in real time. (a, b) Fluorescence channel, Z stack images #5 and #8 from the top, respectively. Nuclei are marked with white arrowheads. (c) Light field image. A 3D image is shown. Scale bars are 100 μM. Embryos were viewed with a 10X objective on a Zeiss LSM 510 confocal microscope equipped with a META detector, and analyzed using LSM510 Image Acquisition software. At least 20 embryos were injected in at least 3 separate experiments.

Techniques Used: In Vitro, Confocal Microscopy, Fluorescence, Microscopy, Software, Injection

9) Product Images from "N-acetyl cysteine reverts the proinflammatory state induced by cigarette smoke extract in lung Calu-3 cells"

Article Title: N-acetyl cysteine reverts the proinflammatory state induced by cigarette smoke extract in lung Calu-3 cells

Journal: Redox Biology

doi: 10.1016/j.redox.2018.03.006

Effects of CSE on mitochondrial ROS levels and Complex I-III activity. A) Confocal microscopy corresponding to mitochondrial ROS (mtROS) levels measured by using MitoSOX at different times (0, 1, 5 and 10 min) in the presence of 100 µg/ml of CSE. Images were taken by using the time series configuration of the LSM 510 confocal microscope, a Plan-Neofluar 100×/1.3 Oil objective, a laser filter of 488 nm, and a LP filter of 560 nm. VIS indicates the visible image of cells. B) Normalized MitoSOX fluoresce values of corrected total cell fluorescence (CTCF) corresponding to the images shown in A. Fluorescence was normalized relative to control cells. C: Mitochondrial NADH-cytochrome c reductase (mCx-I-III) measured in Calu-3 cells incubated with 100 μg/ml CSE (CSE), DMSO or 100 μg/ml CSE + 5 mM NAC (CSE+NAC) for 24 h. Data were expressed as mean ± SEM of three independent experiments (n = 3). * indicates p
Figure Legend Snippet: Effects of CSE on mitochondrial ROS levels and Complex I-III activity. A) Confocal microscopy corresponding to mitochondrial ROS (mtROS) levels measured by using MitoSOX at different times (0, 1, 5 and 10 min) in the presence of 100 µg/ml of CSE. Images were taken by using the time series configuration of the LSM 510 confocal microscope, a Plan-Neofluar 100×/1.3 Oil objective, a laser filter of 488 nm, and a LP filter of 560 nm. VIS indicates the visible image of cells. B) Normalized MitoSOX fluoresce values of corrected total cell fluorescence (CTCF) corresponding to the images shown in A. Fluorescence was normalized relative to control cells. C: Mitochondrial NADH-cytochrome c reductase (mCx-I-III) measured in Calu-3 cells incubated with 100 μg/ml CSE (CSE), DMSO or 100 μg/ml CSE + 5 mM NAC (CSE+NAC) for 24 h. Data were expressed as mean ± SEM of three independent experiments (n = 3). * indicates p

Techniques Used: Activity Assay, Confocal Microscopy, Microscopy, Fluorescence, Incubation

10) Product Images from "A Novel Fluorescence Resonance Energy Transfer Assay Demonstrates that the Human Immunodeficiency Virus Type 1 Pr55Gag I Domain Mediates Gag-Gag Interactions"

Article Title: A Novel Fluorescence Resonance Energy Transfer Assay Demonstrates that the Human Immunodeficiency Virus Type 1 Pr55Gag I Domain Mediates Gag-Gag Interactions

Journal: Journal of Virology

doi: 10.1128/JVI.78.3.1230-1242.2004

Visualization of Gag-Gag FRET by confocal microscopy. Gag-CFP and Gag-YFP were cotransfected in Mel JuSo cells, and images were obtained with a Zeiss LSM 510 confocal microscope equipped with a Meta multichannel detector. Fluorescence excitation was carried out at 458 nm; emission images prepared by linear unmixing techniques for CFP (left panels) and YFP (right panels) are shown. (A) CFP emission image demonstrating weak membrane fluorescence in the indicated region before photobleaching. (B) FRET image indicating efficient plasma membrane Gag-Gag FRET prior to photobleaching of YFP. (C) CFP emission image following photobleaching of YFP in the circled region. Note the increase in CFP emission following YFP photobleaching within the indicated region. (D) FRET image following photobleaching of the indicated area of the cell.
Figure Legend Snippet: Visualization of Gag-Gag FRET by confocal microscopy. Gag-CFP and Gag-YFP were cotransfected in Mel JuSo cells, and images were obtained with a Zeiss LSM 510 confocal microscope equipped with a Meta multichannel detector. Fluorescence excitation was carried out at 458 nm; emission images prepared by linear unmixing techniques for CFP (left panels) and YFP (right panels) are shown. (A) CFP emission image demonstrating weak membrane fluorescence in the indicated region before photobleaching. (B) FRET image indicating efficient plasma membrane Gag-Gag FRET prior to photobleaching of YFP. (C) CFP emission image following photobleaching of YFP in the circled region. Note the increase in CFP emission following YFP photobleaching within the indicated region. (D) FRET image following photobleaching of the indicated area of the cell.

Techniques Used: Confocal Microscopy, Microscopy, Fluorescence

Analysis of Gag-Gag interactions by FRET microscopy and spectral analysis. (A) Gag-CFP and Gag-YFP were cotransfected in Mel JuSo cells, and images were obtained with a Zeiss LSM 510-Meta confocal microscope. The image represents YFP excitation-emission before photobleaching. The arrow indicates the selected plasma membrane region to be bleached. (B) The same cell as that shown in panel A is depicted following photobleaching at 514 nm. (C) Emission scans were obtained from the selected region of interest, with excitation at 458 nm (CFP excitation), before (solid line) and after (dashed line) photobleaching of YFP. Peak emission wavelengths for CFP (secondary peak) and YFP are indicated. (D) Gag-YFP distribution before bleaching. (E) Gag-YFP after photobleaching of indicated region. (F) Spectra obtained before (solid line) and after (dashed line) photobleaching of cells from panels D and E. (G) Cotransfection of Myr −  Gag-YFP and Myr −  Gag-CFP before photobleaching. (H) The same cell as that shown in panel G is depicted after photobleaching of indicated region of the cytoplasm. (I) Spectral analysis of the cell shown in panels G and H.
Figure Legend Snippet: Analysis of Gag-Gag interactions by FRET microscopy and spectral analysis. (A) Gag-CFP and Gag-YFP were cotransfected in Mel JuSo cells, and images were obtained with a Zeiss LSM 510-Meta confocal microscope. The image represents YFP excitation-emission before photobleaching. The arrow indicates the selected plasma membrane region to be bleached. (B) The same cell as that shown in panel A is depicted following photobleaching at 514 nm. (C) Emission scans were obtained from the selected region of interest, with excitation at 458 nm (CFP excitation), before (solid line) and after (dashed line) photobleaching of YFP. Peak emission wavelengths for CFP (secondary peak) and YFP are indicated. (D) Gag-YFP distribution before bleaching. (E) Gag-YFP after photobleaching of indicated region. (F) Spectra obtained before (solid line) and after (dashed line) photobleaching of cells from panels D and E. (G) Cotransfection of Myr − Gag-YFP and Myr − Gag-CFP before photobleaching. (H) The same cell as that shown in panel G is depicted after photobleaching of indicated region of the cytoplasm. (I) Spectral analysis of the cell shown in panels G and H.

Techniques Used: Microscopy, Cotransfection

The I domain mediates Gag-Gag interactions. (A) Gag fusion constructs were expressed in 293T cells and harvested by Dounce homogenization. Whole-cell lysates were analyzed by fluorometry. The intensities of curves were normalized based on the amount of YFP expressed. The YFP-alone emission curve for each pair of constructs was subtracted from the curve of the CFP-YFP combination (i.e., MA-CFP-MA-YFP curve − MA-YFP-alone curve), resulting in an emission peak at 527 nm representing FRET with the background subtracted for each construct. (B) Gag384-CFP and Gag384-YFP were cotransfected in Mel JuSo cells, and images were obtained with a Zeiss LSM 510-Meta confocal microscope. The image represents YFP excitation-emission before photobleaching. (C) The same cell as that shown in panel B following photobleaching of the indicated regions at 514 nm. (D) Emission spectra were obtained from the selected regions of interest before (diamonds) and after (squares) photobleaching. The excitation wavelength for the scan was 458 nm. (E) Gag384(R380,384A)-CFP cotransfected with Gag384(R380,384A)-YFP. The image represents YFP excitation-emission. (F) The same cell as that shown in panel E following photobleaching at 514 nm. (G) Spectral data obtained from the indicated regions of panels E and F before bleaching (diamonds) and after bleaching (squares).
Figure Legend Snippet: The I domain mediates Gag-Gag interactions. (A) Gag fusion constructs were expressed in 293T cells and harvested by Dounce homogenization. Whole-cell lysates were analyzed by fluorometry. The intensities of curves were normalized based on the amount of YFP expressed. The YFP-alone emission curve for each pair of constructs was subtracted from the curve of the CFP-YFP combination (i.e., MA-CFP-MA-YFP curve − MA-YFP-alone curve), resulting in an emission peak at 527 nm representing FRET with the background subtracted for each construct. (B) Gag384-CFP and Gag384-YFP were cotransfected in Mel JuSo cells, and images were obtained with a Zeiss LSM 510-Meta confocal microscope. The image represents YFP excitation-emission before photobleaching. (C) The same cell as that shown in panel B following photobleaching of the indicated regions at 514 nm. (D) Emission spectra were obtained from the selected regions of interest before (diamonds) and after (squares) photobleaching. The excitation wavelength for the scan was 458 nm. (E) Gag384(R380,384A)-CFP cotransfected with Gag384(R380,384A)-YFP. The image represents YFP excitation-emission. (F) The same cell as that shown in panel E following photobleaching at 514 nm. (G) Spectral data obtained from the indicated regions of panels E and F before bleaching (diamonds) and after bleaching (squares).

Techniques Used: Construct, Homogenization, Microscopy

11) Product Images from "Utilizing HaloTag Technology to Track the Fate of PCSK9 from Intracellular vs. Extracellular Sources"

Article Title: Utilizing HaloTag Technology to Track the Fate of PCSK9 from Intracellular vs. Extracellular Sources

Journal: Current Chemical Genomics

doi: 10.2174/1875397301206010038

PCSK9-HT is labeled with HaloTag ligands TMR and Alexa Fluor 488 . A Huh7 stable cell line expressing C-terminal PCSK9-HaloTag fusion protein was established and verified with western blot ( A , clone 14-13-E4). The expression level of PCSK9-HT is about 2-fold of the endogenous PCSK9 level. B . Purified PCSK9-HT inhibited LDL uptake in a dose dependent manner in HepG2 cells. The de-creased potency of PCSK9-HT (red) compared to wt PCSK9 (blue) is likely due to protein impurity. C . 14-13-E4 cells were labeled with 0.2 µM Alexa Fluor 488 overnight, followed by TMR labeling (5 µM) for 15 min. The unbound ligand was washed off before the second label-ing or image capture. Confocal images were taken with a Zeiss LSM 510 confocal microscope with appropriate fluorescence filters. Intracel-lular PCSK9 (red) and internalized PCSK9 (green) showed different staining patterns.
Figure Legend Snippet: PCSK9-HT is labeled with HaloTag ligands TMR and Alexa Fluor 488 . A Huh7 stable cell line expressing C-terminal PCSK9-HaloTag fusion protein was established and verified with western blot ( A , clone 14-13-E4). The expression level of PCSK9-HT is about 2-fold of the endogenous PCSK9 level. B . Purified PCSK9-HT inhibited LDL uptake in a dose dependent manner in HepG2 cells. The de-creased potency of PCSK9-HT (red) compared to wt PCSK9 (blue) is likely due to protein impurity. C . 14-13-E4 cells were labeled with 0.2 µM Alexa Fluor 488 overnight, followed by TMR labeling (5 µM) for 15 min. The unbound ligand was washed off before the second label-ing or image capture. Confocal images were taken with a Zeiss LSM 510 confocal microscope with appropriate fluorescence filters. Intracel-lular PCSK9 (red) and internalized PCSK9 (green) showed different staining patterns.

Techniques Used: Labeling, Stable Transfection, Expressing, Western Blot, Purification, Microscopy, Fluorescence, Staining

12) Product Images from "Detection of Human Immunodeficiency Virus Type 1 Nef and CD4 Physical Interaction in Living Human Cells by Using Bioluminescence Resonance Energy Transfer"

Article Title: Detection of Human Immunodeficiency Virus Type 1 Nef and CD4 Physical Interaction in Living Human Cells by Using Bioluminescence Resonance Energy Transfer

Journal: Journal of Virology

doi: 10.1128/JVI.79.13.8629-8636.2005

The different fusion proteins are correctly addressed in HEK-293 cells. (A) HEK-293 cells (6.5 × 10 4 ) were cotransfected with 1 μg pHIS/β2Adr-EYFP, pCi-Neo/Nef-Rluc, pCi-Neo/Nef-EYFP, pcDNA3/CD4-EYFP, or pCi-Neo/CD4 414AA-EYFP. Twenty-four hours posttransfection, the cells expressing proteins fused to EYFP were fixed and observed with a Zeiss LSM 510 confocal microscope. The cells producing Nef-Rluc were fixed and permeabilized. The Nef protein was primary marked with maTG020 and labeled with an FITC-conjugated goat anti-mouse immunoglobulin G antibody. (B) To assess the colocalization of Nef or Nef-Rluc with the two CD4 variants at the plasma membrane, HEK-293 cells were cotransfected with 0.5 μg pCi-Neo/Nef-Lai or pCi-Neo/Nef-Rluc and the same amount of pcDNA3/CD4 or pCML/CD4 414AA. Forty-eight hours posttransfection, the cells were fixed and permeabilized. The Nef proteins were marked as previously. The CD4 molecules were labeled with the RPA-T4-PC5 antibody. The colocalization panels represent the overlay of CD4-PC5 and Nef-FITC fluorescences. Bars correspond to 10 μm.
Figure Legend Snippet: The different fusion proteins are correctly addressed in HEK-293 cells. (A) HEK-293 cells (6.5 × 10 4 ) were cotransfected with 1 μg pHIS/β2Adr-EYFP, pCi-Neo/Nef-Rluc, pCi-Neo/Nef-EYFP, pcDNA3/CD4-EYFP, or pCi-Neo/CD4 414AA-EYFP. Twenty-four hours posttransfection, the cells expressing proteins fused to EYFP were fixed and observed with a Zeiss LSM 510 confocal microscope. The cells producing Nef-Rluc were fixed and permeabilized. The Nef protein was primary marked with maTG020 and labeled with an FITC-conjugated goat anti-mouse immunoglobulin G antibody. (B) To assess the colocalization of Nef or Nef-Rluc with the two CD4 variants at the plasma membrane, HEK-293 cells were cotransfected with 0.5 μg pCi-Neo/Nef-Lai or pCi-Neo/Nef-Rluc and the same amount of pcDNA3/CD4 or pCML/CD4 414AA. Forty-eight hours posttransfection, the cells were fixed and permeabilized. The Nef proteins were marked as previously. The CD4 molecules were labeled with the RPA-T4-PC5 antibody. The colocalization panels represent the overlay of CD4-PC5 and Nef-FITC fluorescences. Bars correspond to 10 μm.

Techniques Used: Expressing, Microscopy, Labeling, Recombinase Polymerase Amplification

13) Product Images from "Phosphorylation of Amyloid Precursor Protein (APP) at Thr668 Regulates the Nuclear Translocation of the APP Intracellular Domain and Induces Neurodegeneration"

Article Title: Phosphorylation of Amyloid Precursor Protein (APP) at Thr668 Regulates the Nuclear Translocation of the APP Intracellular Domain and Induces Neurodegeneration

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.02393-05

pAPP T668 , GSK-3β, and p-tau are upregulated in Tg2576 mouse brains. The Tg2576 mouse brains ( n = 5) fixed in 10% neutral buffered formalin for 48 h were dehydrated and embedded in paraffin. The fluorescent immunohistochemistry was performed with appropriate primary antibodies for 2 h and visualized using Cy3-conjugated or fluorescein isothiocyanate-conjugated secondary antibody (Jackson, West Grove, Pennsylvania). DAPI counterstaining was performed. Images were collected using the LSM 510 program on a Zeiss confocal microscope. (a) The immunoreactivities of pAPP T668 (green) and GSK-3β (red) were examined in CA1 and CA3 of Tg2576 mouse brains (Tg2576) and WT mouse brains. DAPI staining shows the location of the nucleus (blue). Bar, 50 μm. Magnification, ×353.44. (b) Immunoreactivities of pAPP T668 (green) and p-tau (red) were examined in Tg2576 mice and WT mouse brains. Bar, 50 μm. Magnification, ×353.44. (c, d) Protein levels of AICD and CTFs were examined by immunoblotting using C9 antibody which recognizes the last 9 amino acids of APP (c) and anti-pAPP (T668) antibody (d) in the total lysates of cortex or hippocampus in Tg2576 mice and WT mouse brains. Densitometric analysis was done, and data represent the means ± standard errors of results from three or six separate experiments. **, P
Figure Legend Snippet: pAPP T668 , GSK-3β, and p-tau are upregulated in Tg2576 mouse brains. The Tg2576 mouse brains ( n = 5) fixed in 10% neutral buffered formalin for 48 h were dehydrated and embedded in paraffin. The fluorescent immunohistochemistry was performed with appropriate primary antibodies for 2 h and visualized using Cy3-conjugated or fluorescein isothiocyanate-conjugated secondary antibody (Jackson, West Grove, Pennsylvania). DAPI counterstaining was performed. Images were collected using the LSM 510 program on a Zeiss confocal microscope. (a) The immunoreactivities of pAPP T668 (green) and GSK-3β (red) were examined in CA1 and CA3 of Tg2576 mouse brains (Tg2576) and WT mouse brains. DAPI staining shows the location of the nucleus (blue). Bar, 50 μm. Magnification, ×353.44. (b) Immunoreactivities of pAPP T668 (green) and p-tau (red) were examined in Tg2576 mice and WT mouse brains. Bar, 50 μm. Magnification, ×353.44. (c, d) Protein levels of AICD and CTFs were examined by immunoblotting using C9 antibody which recognizes the last 9 amino acids of APP (c) and anti-pAPP (T668) antibody (d) in the total lysates of cortex or hippocampus in Tg2576 mice and WT mouse brains. Densitometric analysis was done, and data represent the means ± standard errors of results from three or six separate experiments. **, P

Techniques Used: Immunohistochemistry, Microscopy, Staining, Mouse Assay

14) Product Images from "Association of Human DEAD Box Protein DDX1 with a Cleavage Stimulation Factor Involved in 3?-End Processing of Pre-mRNA V⃞"

Article Title: Association of Human DEAD Box Protein DDX1 with a Cleavage Stimulation Factor Involved in 3?-End Processing of Pre-mRNA V⃞

Journal: Molecular Biology of the Cell

doi:

DDX1 foci do not accumulate nascent RNA. (A) HeLa cells were incubated with FU for 15 min and stained with anti-DDX1 antibody (2923) and anti-bromodeoxyuridine antibody (FU). The arrowhead indicates a DDX1 focus. (B) The staining intensities of the region through a DDX1 foci (highlighted by the arrow in the bottom left panel) were profiled with the use of the Zeiss LSM 510 image software. The green line represents FU intensity, whereas the red line represents DDX1 intensity. Bar, 5 μm.
Figure Legend Snippet: DDX1 foci do not accumulate nascent RNA. (A) HeLa cells were incubated with FU for 15 min and stained with anti-DDX1 antibody (2923) and anti-bromodeoxyuridine antibody (FU). The arrowhead indicates a DDX1 focus. (B) The staining intensities of the region through a DDX1 foci (highlighted by the arrow in the bottom left panel) were profiled with the use of the Zeiss LSM 510 image software. The green line represents FU intensity, whereas the red line represents DDX1 intensity. Bar, 5 μm.

Techniques Used: Incubation, Staining, Software

Immunofluorescence labeling of DDX1. HeLa cells were immunolabeled with anti-DDX1 polyclonal antibody (2923). (A) General staining pattern of DDX1 and its distribution relative to chromatin (DAPI). DDX1 is widely expressed in the nucleus, where it is found in both foci and nucleoplasm. The DDX1 signal has a punctate appearance in both the nucleoplasm and cytoplasm. Nuclear foci are shown in red in the merged (DDX1 + DAPI) image. The cells were observed with a Zeiss Axioplan II microscope equipped with a cooled, charge-coupled device camera; the image collected was deconvolved with the use of Softworks 2.5 software. (B) DDX1 foci are often visible by digital interference contrast (DIC) (indicated by the arrowhead). The image was collected with a Zeiss LSM 510 confocal laser scanning microscope. (C) Anti-DDX1 antibody is specifically competed by purified DDX1. Either anti-DDX1 polyclonal antibody (2923) or anti-ALDH polyclonal antibody was incubated overnight with either 0 or 5 μg of nitrocellulose-bound purifed DDX1 protein. The unbound fraction was then used to indirectly label HeLa cells. The image was collected with a Zeiss Axioplan II microscope. The DDX1 signal is greatly reduced after antibody adsorption to 5 μg of DDX1 protein, whereas the ALDH signal is unaffected. Bar, 5 μm.
Figure Legend Snippet: Immunofluorescence labeling of DDX1. HeLa cells were immunolabeled with anti-DDX1 polyclonal antibody (2923). (A) General staining pattern of DDX1 and its distribution relative to chromatin (DAPI). DDX1 is widely expressed in the nucleus, where it is found in both foci and nucleoplasm. The DDX1 signal has a punctate appearance in both the nucleoplasm and cytoplasm. Nuclear foci are shown in red in the merged (DDX1 + DAPI) image. The cells were observed with a Zeiss Axioplan II microscope equipped with a cooled, charge-coupled device camera; the image collected was deconvolved with the use of Softworks 2.5 software. (B) DDX1 foci are often visible by digital interference contrast (DIC) (indicated by the arrowhead). The image was collected with a Zeiss LSM 510 confocal laser scanning microscope. (C) Anti-DDX1 antibody is specifically competed by purified DDX1. Either anti-DDX1 polyclonal antibody (2923) or anti-ALDH polyclonal antibody was incubated overnight with either 0 or 5 μg of nitrocellulose-bound purifed DDX1 protein. The unbound fraction was then used to indirectly label HeLa cells. The image was collected with a Zeiss Axioplan II microscope. The DDX1 signal is greatly reduced after antibody adsorption to 5 μg of DDX1 protein, whereas the ALDH signal is unaffected. Bar, 5 μm.

Techniques Used: Immunofluorescence, Labeling, Immunolabeling, Staining, Microscopy, Software, Laser-Scanning Microscopy, Purification, Incubation, Adsorption

15) Product Images from "Corynebacterium diphtheriae employs specific minor pilins to target human pharyngeal epithelial cells"

Article Title: Corynebacterium diphtheriae employs specific minor pilins to target human pharyngeal epithelial cells

Journal: Molecular microbiology

doi: 10.1111/j.1365-2958.2007.05630.x

Immunofluorescent detection of SpaB and SpaC pilins on the bacterial surface. Corynebacteria were stained with a specific antibody against SpaA (α-SpaA), SpaB (α-SpaB) or SpaC (α-SpaC) and AlexaFluor 488 chicken anti-rabbit IgG. Shown are the fluorescent, the Nomarski DIC and the merged images of strain Δ srtB–F (A), its isogenic derivative Δ spaA (B) or its isogenic derivative Δ spaA transformed with a plasmid expressing the SpaA protein mutated at the K190 in the pilin motif (C). The samples were observed on a Zeiss LSM 510 confocal microscope.
Figure Legend Snippet: Immunofluorescent detection of SpaB and SpaC pilins on the bacterial surface. Corynebacteria were stained with a specific antibody against SpaA (α-SpaA), SpaB (α-SpaB) or SpaC (α-SpaC) and AlexaFluor 488 chicken anti-rabbit IgG. Shown are the fluorescent, the Nomarski DIC and the merged images of strain Δ srtB–F (A), its isogenic derivative Δ spaA (B) or its isogenic derivative Δ spaA transformed with a plasmid expressing the SpaA protein mutated at the K190 in the pilin motif (C). The samples were observed on a Zeiss LSM 510 confocal microscope.

Techniques Used: Staining, Transformation Assay, Plasmid Preparation, Expressing, Microscopy

Anchoring of minor pilins SpaB and SpaC required the conserved LPXTG motif. Strain Δ srtB–F with a deletion of spaA , spaB or spaC gene was transformed with a plasmid expressing the wild-type or mutated SpaA, SpaB or SpaC respectively. Corynebacteria were stained with a specific antibody against SpaA (α-SpaA), SpaB (α-SpaB) or SpaC (α-SpaC) and AlexaFluor 488 chicken anti-rabbit IgG. The samples were observed on a Zeiss LSM 510 confocal microscope. Only fluorescent images are shown.
Figure Legend Snippet: Anchoring of minor pilins SpaB and SpaC required the conserved LPXTG motif. Strain Δ srtB–F with a deletion of spaA , spaB or spaC gene was transformed with a plasmid expressing the wild-type or mutated SpaA, SpaB or SpaC respectively. Corynebacteria were stained with a specific antibody against SpaA (α-SpaA), SpaB (α-SpaB) or SpaC (α-SpaC) and AlexaFluor 488 chicken anti-rabbit IgG. The samples were observed on a Zeiss LSM 510 confocal microscope. Only fluorescent images are shown.

Techniques Used: Transformation Assay, Plasmid Preparation, Expressing, Staining, Microscopy

16) Product Images from "Expression and subcellular distribution of UNC119a, a protein partner of transducin ? subunit in rod photoreceptors"

Article Title: Expression and subcellular distribution of UNC119a, a protein partner of transducin ? subunit in rod photoreceptors

Journal: Cellular signalling

doi: 10.1016/j.cellsig.2012.10.005

Gα t1 and UNC119a co-localize following light-dependent translocation of transducin Cryosections of mouse retinas were obtained from dark- (DA) and light-adapted (LA: 45 min, 500 lux) Gα t1 +/− ] (green) and a rabbit anti-Gα t1 antibody K-20 (SCBT) (red). The staining was visualized using goat anti-rabbit AlexaFluor 568 and goat anti-mouse AlexaFluor 488 secondary antibodies under a Zeiss LSM 510 confocal microscope. Bar – 20 μm. OS – outer segment, IS – inner segment, ONL – outer nuclear layer, OPL – outer plexiform layer.
Figure Legend Snippet: Gα t1 and UNC119a co-localize following light-dependent translocation of transducin Cryosections of mouse retinas were obtained from dark- (DA) and light-adapted (LA: 45 min, 500 lux) Gα t1 +/− ] (green) and a rabbit anti-Gα t1 antibody K-20 (SCBT) (red). The staining was visualized using goat anti-rabbit AlexaFluor 568 and goat anti-mouse AlexaFluor 488 secondary antibodies under a Zeiss LSM 510 confocal microscope. Bar – 20 μm. OS – outer segment, IS – inner segment, ONL – outer nuclear layer, OPL – outer plexiform layer.

Techniques Used: Translocation Assay, Staining, Microscopy

17) Product Images from "Positive Feedback Regulation between Phospholipase D and Wnt Signaling Promotes Wnt-Driven Anchorage-Independent Growth of Colorectal Cancer Cells"

Article Title: Positive Feedback Regulation between Phospholipase D and Wnt Signaling Promotes Wnt-Driven Anchorage-Independent Growth of Colorectal Cancer Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0012109

LiCl increases expression of PLD isozymes in vivo . (A) Mice were intravenously injected with LiCl, as described in “ Materials and Methods ”. Lysates from various tissues were immunoprecipitated and immunoblotted with antibody to PLD recognizing both PLD1 and PLD2 (left panel). Protein levels were analyzed by immunoblot using the indicated antibodies. Histograms show relative protein levels of PLD1 and PLD2, which are normalized to the corresponding α-tubulin values (right panel). (B) Paraffin sections of colon tissues were subjected to immunofluorescence analyses using anti-β-catenin (Alexa fluor 488; green) and PLD (Alexa fluor 555; red) antibody. Tissues were monitored using Zeiss LSM 510 confocal microscope. Microscopy fields were observed at × 650 magnification. Data are representative of three independent experiments.
Figure Legend Snippet: LiCl increases expression of PLD isozymes in vivo . (A) Mice were intravenously injected with LiCl, as described in “ Materials and Methods ”. Lysates from various tissues were immunoprecipitated and immunoblotted with antibody to PLD recognizing both PLD1 and PLD2 (left panel). Protein levels were analyzed by immunoblot using the indicated antibodies. Histograms show relative protein levels of PLD1 and PLD2, which are normalized to the corresponding α-tubulin values (right panel). (B) Paraffin sections of colon tissues were subjected to immunofluorescence analyses using anti-β-catenin (Alexa fluor 488; green) and PLD (Alexa fluor 555; red) antibody. Tissues were monitored using Zeiss LSM 510 confocal microscope. Microscopy fields were observed at × 650 magnification. Data are representative of three independent experiments.

Techniques Used: Expressing, In Vivo, Mouse Assay, Injection, Immunoprecipitation, Immunofluorescence, Microscopy

18) Product Images from "Primary Lung Dendritic Cell Cultures to Assess Efficacy of Spectinamide-1599 Against Intracellular Mycobacterium tuberculosis"

Article Title: Primary Lung Dendritic Cell Cultures to Assess Efficacy of Spectinamide-1599 Against Intracellular Mycobacterium tuberculosis

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2018.01895

Localization and quantification of intracellular Mtb bacilli in lung derived DCs. Lung derived DC cultures were infected with Mycobacterium tuberculosis (H37Ra strain) as explained in Section “Materials and Methods.” Cells were washed and cultured with cRPMI media (without antibiotics) containing GM-CSF as in Figure 1 . Acid fast positive bacilli within cells were visualized using a confocal ZEISS LSM 510 microscope after staining of lung DC cells by the acid fast staining method with Sybr Gold (green). (A) Intracellular H37Ra bacilli in lung dendritic cell cultures after 7 days of infection. (B) Magnification of picture in (A) , showing cells containing one, two or many bacilli. (C) Demonstration of intracellular location of bacilli in a confocal imaging Z -stack of cells scanned at 0.2 μm steps. (D) The Mtb bacilli burden in lung dendritic cell homogenates infected for 1, 7, 15, and 30 days (black bars) in similar cultures of lung DCs infected with Mtb as in (A) . The Mtb bacilli burden in supernatants from each cell culture is represented in gray bars. The number of colony forming units (CFU) is expressed as log 10 CFU. The results suggest that almost all bacteria are located intracellularly, and that there is no significant change in the number of intracellular bacteria over the observation period. The dotted line represents the detection limit of the assay. Representative data from more than three experiments in similar conditions.
Figure Legend Snippet: Localization and quantification of intracellular Mtb bacilli in lung derived DCs. Lung derived DC cultures were infected with Mycobacterium tuberculosis (H37Ra strain) as explained in Section “Materials and Methods.” Cells were washed and cultured with cRPMI media (without antibiotics) containing GM-CSF as in Figure 1 . Acid fast positive bacilli within cells were visualized using a confocal ZEISS LSM 510 microscope after staining of lung DC cells by the acid fast staining method with Sybr Gold (green). (A) Intracellular H37Ra bacilli in lung dendritic cell cultures after 7 days of infection. (B) Magnification of picture in (A) , showing cells containing one, two or many bacilli. (C) Demonstration of intracellular location of bacilli in a confocal imaging Z -stack of cells scanned at 0.2 μm steps. (D) The Mtb bacilli burden in lung dendritic cell homogenates infected for 1, 7, 15, and 30 days (black bars) in similar cultures of lung DCs infected with Mtb as in (A) . The Mtb bacilli burden in supernatants from each cell culture is represented in gray bars. The number of colony forming units (CFU) is expressed as log 10 CFU. The results suggest that almost all bacteria are located intracellularly, and that there is no significant change in the number of intracellular bacteria over the observation period. The dotted line represents the detection limit of the assay. Representative data from more than three experiments in similar conditions.

Techniques Used: Derivative Assay, Infection, Cell Culture, Microscopy, Staining, Imaging

19) Product Images from "Arginine Methylation of the RGG Box Does Not Appear To Regulate ICP27 Import during Herpes Simplex Virus Infection ▿"

Article Title: Arginine Methylation of the RGG Box Does Not Appear To Regulate ICP27 Import during Herpes Simplex Virus Infection ▿

Journal: Journal of Virology

doi: 10.1128/JVI.00679-11

FRAP analysis of import of ICP27 under conditions of hypomethylation. (A) HeLa cells were infected with vN-YFP-ICP27 at an MOI of 5. At 6 h after infection, photobleaching was performed using living infected cells, a Zeiss LSM 510 confocal microscope, and FRAP software. The entire circled nucleus in each cell was photobleached. The laser was set at 100% power, and bleaching was performed using the 514-nm laser line. The acousto-optical tunable filter (AOTF) was set at 100%, and 50 iterations of the procedure were performed. Images were captured every 125 s after bleaching. (B) The percentages of fluorescence recovery corresponding to the indicated time points for the cell marked by the pink arrow in the +AdOx field and for the cell marked by the red arrow in the no-AdOx field are shown. Percent fluorescence recovery is the amount of fluorescence after photobleaching divided by the amount of fluorescence before photobleaching multiplied by 100 according to the formula ( Y / X ) × 100 = % recovery, where the percentage of fluorescence lost due to photobleaching is represented by X and the amount of fluorescence that returned to the bleached area is represented by Y .
Figure Legend Snippet: FRAP analysis of import of ICP27 under conditions of hypomethylation. (A) HeLa cells were infected with vN-YFP-ICP27 at an MOI of 5. At 6 h after infection, photobleaching was performed using living infected cells, a Zeiss LSM 510 confocal microscope, and FRAP software. The entire circled nucleus in each cell was photobleached. The laser was set at 100% power, and bleaching was performed using the 514-nm laser line. The acousto-optical tunable filter (AOTF) was set at 100%, and 50 iterations of the procedure were performed. Images were captured every 125 s after bleaching. (B) The percentages of fluorescence recovery corresponding to the indicated time points for the cell marked by the pink arrow in the +AdOx field and for the cell marked by the red arrow in the no-AdOx field are shown. Percent fluorescence recovery is the amount of fluorescence after photobleaching divided by the amount of fluorescence before photobleaching multiplied by 100 according to the formula ( Y / X ) × 100 = % recovery, where the percentage of fluorescence lost due to photobleaching is represented by X and the amount of fluorescence that returned to the bleached area is represented by Y .

Techniques Used: Infection, Microscopy, Software, Fluorescence

20) Product Images from "Enterococcus faecalis Infection Causes Inflammation, Intracellular Oxphos-Independent ROS Production, and DNA Damage in Human Gastric Cancer Cells"

Article Title: Enterococcus faecalis Infection Causes Inflammation, Intracellular Oxphos-Independent ROS Production, and DNA Damage in Human Gastric Cancer Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0063147

E. faecalis infection induced intracellular ROS production. MKN74 cells infected with E. faecalis for 30 minutes at MOI50. (A) Representative fluorescence microscope image of MKN74 cells stained with ROS detecting probes (green) and superoxide detecting probes (orange) Scale bars = 50 µm. (B) Quantification of fluorescence intensity using the LSM 510 software. A statistically significant increased intracellular production of ROS (p
Figure Legend Snippet: E. faecalis infection induced intracellular ROS production. MKN74 cells infected with E. faecalis for 30 minutes at MOI50. (A) Representative fluorescence microscope image of MKN74 cells stained with ROS detecting probes (green) and superoxide detecting probes (orange) Scale bars = 50 µm. (B) Quantification of fluorescence intensity using the LSM 510 software. A statistically significant increased intracellular production of ROS (p

Techniques Used: Infection, Fluorescence, Microscopy, Staining, Software

21) Product Images from "Merkel Cell Polyomavirus Small T Antigen Mediates Microtubule Destabilization To Promote Cell Motility and Migration"

Article Title: Merkel Cell Polyomavirus Small T Antigen Mediates Microtubule Destabilization To Promote Cell Motility and Migration

Journal: Journal of Virology

doi: 10.1128/JVI.02317-14

MCPyV ST expression leads to the differential expression of proteins involved in microtubule-associated cytoskeletal organization and dynamics. (A) (i) i293-ST cells were grown in DMEM with R0K0 and induced (IN) for 24 h or grown in DMEM with R6K4 and remained uninduced (UN). Cell lysates were analyzed by immunoblotting with a FLAG-specific antibody. (ii) To confirm that induced levels of MCPyV ST in i293-ST cells are representative of ST expression in the MCPyV-positive MCC cell lines, immunoblotting was performed using an MCPyV T-specific antibody comparing cell lysates from 1 × 10 5 cells of uninduced and induced i293-ST, MCC13, and MKL-1 cells. (B) i293-ST cells remained uninduced or were incubated for either 24 or 48 h in the presence of doxycycline hyclate. After induction, cell lysates were analyzed by immunoblotting using a FLAG-specific antibody and a range of microtubule-associated-specific antibodies highlighted by quantitative proteomic analysis. GAPDH was used as a measure of equal loading. (C) FFPE sections of a primary MCC tumor were stained with stathmin- and CK20-specific antibodies or an isotype negative control. After washing, sections were incubated with Alexa Fluor-labeled secondary antibodies. Nuclear staining was performed with bis-benzimide. Slides were then analyzed using a Zeiss LSM 510 confocal laser scanning microscope. (D) FFPE sections of two additional primary MCC tumors were stained with stathmin-, MCPyV LT-, and CK20-specific antibodies or an isotype negative control. After washing, sections were incubated with Alexa Fluor-labeled secondary antibodies. Nuclear staining was labeled using bis-benzimide. Slides were then analyzed using a Zeiss LSM 510 confocal laser scanning microscope.
Figure Legend Snippet: MCPyV ST expression leads to the differential expression of proteins involved in microtubule-associated cytoskeletal organization and dynamics. (A) (i) i293-ST cells were grown in DMEM with R0K0 and induced (IN) for 24 h or grown in DMEM with R6K4 and remained uninduced (UN). Cell lysates were analyzed by immunoblotting with a FLAG-specific antibody. (ii) To confirm that induced levels of MCPyV ST in i293-ST cells are representative of ST expression in the MCPyV-positive MCC cell lines, immunoblotting was performed using an MCPyV T-specific antibody comparing cell lysates from 1 × 10 5 cells of uninduced and induced i293-ST, MCC13, and MKL-1 cells. (B) i293-ST cells remained uninduced or were incubated for either 24 or 48 h in the presence of doxycycline hyclate. After induction, cell lysates were analyzed by immunoblotting using a FLAG-specific antibody and a range of microtubule-associated-specific antibodies highlighted by quantitative proteomic analysis. GAPDH was used as a measure of equal loading. (C) FFPE sections of a primary MCC tumor were stained with stathmin- and CK20-specific antibodies or an isotype negative control. After washing, sections were incubated with Alexa Fluor-labeled secondary antibodies. Nuclear staining was performed with bis-benzimide. Slides were then analyzed using a Zeiss LSM 510 confocal laser scanning microscope. (D) FFPE sections of two additional primary MCC tumors were stained with stathmin-, MCPyV LT-, and CK20-specific antibodies or an isotype negative control. After washing, sections were incubated with Alexa Fluor-labeled secondary antibodies. Nuclear staining was labeled using bis-benzimide. Slides were then analyzed using a Zeiss LSM 510 confocal laser scanning microscope.

Techniques Used: Expressing, Incubation, Formalin-fixed Paraffin-Embedded, Staining, Negative Control, Labeling, Laser-Scanning Microscopy

22) Product Images from "Binding and activation of host plasminogen on the surface of Francisella tularensis"

Article Title: Binding and activation of host plasminogen on the surface of Francisella tularensis

Journal: BMC Microbiology

doi: 10.1186/1471-2180-10-76

PLG binds to the outer envelope of FT . Laser scanning confocal microscopy of PLG-associated FTLVS was performed as described in
Figure Legend Snippet: PLG binds to the outer envelope of FT . Laser scanning confocal microscopy of PLG-associated FTLVS was performed as described in "Materials and Methods". Bound huPLG ligand was detected using sheep anti-human PLG antibody followed by incubation with Dylight-488 conjugated donkey, anti-sheep/goat IgG secondary antibody. Samples were visualized using a Zeiss LSM 510 confocal microscope.

Techniques Used: Confocal Microscopy, Incubation, Microscopy

23) Product Images from "Autophagy Regulates Lipolysis and Cell Survival Through Lipid Droplet Degradation in Androgen Sensitive Prostate Cancer Cells"

Article Title: Autophagy Regulates Lipolysis and Cell Survival Through Lipid Droplet Degradation in Androgen Sensitive Prostate Cancer Cells

Journal: The Prostate

doi: 10.1002/pros.22489

Autophagosomes colocalize with LDs in LNCaP cells during androgen deprivation LNCaP.EGFP-LC3 cells were incubated for 3 days in indicated medium, (a) CM, (d) CFM; (g) CFM+R1881, and (j) CFM+Baf A1, CFM plus BAF A1 for 12 hours. Cells were fixed, stained with HCSLipidTOX Red, and observed with a Zeiss LSM 510 confocal microscope. Panels of EGFP.LC3 (green), of LipidTOX (red) and merged are as indicated. Colocalization of autophagic vesicles (AVs; green) and lipid droplets (red) were yellow dots shown in the merged panels. In CM, EGFP-LC3 was mainly cytosolic and there were abundant LDs in cells (panels a and b). There was almost no colocalization of EGFP.LC3 and LDs in these cells (panel c; column 1 in panel n). However, when cells were cultured in CFM, which lacked androgen, the EGFP.LC3 molecules translocated to green punctate structures, presumed AVs (panel d; column 2 in panel m), which partially colocalized with the red LDs (panels f; column 2 in panel n). The effect of androgen deprivation could be reversed by adding back R1881 (panels g to I; column 3 in panels m and n). In addition, blocking the autophagic flux by Baf A 1 resulted in a greater accumulation of AVs (panel j; column 4 in panel m) and LDs (panel k) and the number of colocalized AVs and LDs (panel l; column 4 in panel n), indicating that Baf A1 restricted intracellular LD degradation in LNCaP cells. Panel o showed a representative image of a cell filled with AVs partially colocalized with LDs, and a magnified image of a LD enwrapped by AVs (inset).
Figure Legend Snippet: Autophagosomes colocalize with LDs in LNCaP cells during androgen deprivation LNCaP.EGFP-LC3 cells were incubated for 3 days in indicated medium, (a) CM, (d) CFM; (g) CFM+R1881, and (j) CFM+Baf A1, CFM plus BAF A1 for 12 hours. Cells were fixed, stained with HCSLipidTOX Red, and observed with a Zeiss LSM 510 confocal microscope. Panels of EGFP.LC3 (green), of LipidTOX (red) and merged are as indicated. Colocalization of autophagic vesicles (AVs; green) and lipid droplets (red) were yellow dots shown in the merged panels. In CM, EGFP-LC3 was mainly cytosolic and there were abundant LDs in cells (panels a and b). There was almost no colocalization of EGFP.LC3 and LDs in these cells (panel c; column 1 in panel n). However, when cells were cultured in CFM, which lacked androgen, the EGFP.LC3 molecules translocated to green punctate structures, presumed AVs (panel d; column 2 in panel m), which partially colocalized with the red LDs (panels f; column 2 in panel n). The effect of androgen deprivation could be reversed by adding back R1881 (panels g to I; column 3 in panels m and n). In addition, blocking the autophagic flux by Baf A 1 resulted in a greater accumulation of AVs (panel j; column 4 in panel m) and LDs (panel k) and the number of colocalized AVs and LDs (panel l; column 4 in panel n), indicating that Baf A1 restricted intracellular LD degradation in LNCaP cells. Panel o showed a representative image of a cell filled with AVs partially colocalized with LDs, and a magnified image of a LD enwrapped by AVs (inset).

Techniques Used: Incubation, Staining, Microscopy, Cell Culture, Blocking Assay

Androgen deprivation induces autophagy in LNCaP cells, which can be reversed by autophagy inhibitors, 3-MA and Bafilomycin A1 (Baf A1) A. LNCaP.EGFP-LC3 cells (LNCaP cells stably transfected with pEGFP.LC3) were incubated for 2 days in indicated medium, (a) CM, complete medium; (b) CFM, charcoal-filtered fetal bovine serum medium; (c) CFM+R1881, CFM plus R1881; and (d) CFM+3MA, CFM plus 3-methyladenine (3-MA). Cells were mounted in coverslips and observed with a Zeiss LSM 510 confocal microscope. B. Numbers of cells and puncta were counted from 10 random visual fields for each group. Average numbers of puncta per cell were plotted in the graph. C. Blocking autophagy by 3-MA, an inhibitor of the initial stage of autophagy, induced cell death in LNCaP cells cultured in CFM by day 4, as analyzed by MTT assay. D. Induction of autophagy was confirmed in LNCAP cells treated with CFM. Immunoblot analysis showed that treatment of Baf A1, a blocker of the autolysosome formation in the final stage of autophagy, in cells cultured in CFM resulted in greater increase of LC3II (lane 5) compared to cells cultured in CM (lane 4), indicating that androgen deprivation induced autophagy. CDX, an AR inhibitor, showed no effect on LC3 II level and thus autophagy in LNCaP cells cultured in CM (lane 3), whereas combinational treatment of CDX and Baf A1 showed moderate increase of LC3-II, suggesting that the endogenous autophagy is inhibited by BAf A1. E. Time- dependent activation of autophagy in LNCaP.EGFP-LC3 cells cultured in medium CFM. Immunoblot analysis showed that the level of EGFP-LC3II (45 kDa) was increased in a time-dependent manner in cells grown in CFM.
Figure Legend Snippet: Androgen deprivation induces autophagy in LNCaP cells, which can be reversed by autophagy inhibitors, 3-MA and Bafilomycin A1 (Baf A1) A. LNCaP.EGFP-LC3 cells (LNCaP cells stably transfected with pEGFP.LC3) were incubated for 2 days in indicated medium, (a) CM, complete medium; (b) CFM, charcoal-filtered fetal bovine serum medium; (c) CFM+R1881, CFM plus R1881; and (d) CFM+3MA, CFM plus 3-methyladenine (3-MA). Cells were mounted in coverslips and observed with a Zeiss LSM 510 confocal microscope. B. Numbers of cells and puncta were counted from 10 random visual fields for each group. Average numbers of puncta per cell were plotted in the graph. C. Blocking autophagy by 3-MA, an inhibitor of the initial stage of autophagy, induced cell death in LNCaP cells cultured in CFM by day 4, as analyzed by MTT assay. D. Induction of autophagy was confirmed in LNCAP cells treated with CFM. Immunoblot analysis showed that treatment of Baf A1, a blocker of the autolysosome formation in the final stage of autophagy, in cells cultured in CFM resulted in greater increase of LC3II (lane 5) compared to cells cultured in CM (lane 4), indicating that androgen deprivation induced autophagy. CDX, an AR inhibitor, showed no effect on LC3 II level and thus autophagy in LNCaP cells cultured in CM (lane 3), whereas combinational treatment of CDX and Baf A1 showed moderate increase of LC3-II, suggesting that the endogenous autophagy is inhibited by BAf A1. E. Time- dependent activation of autophagy in LNCaP.EGFP-LC3 cells cultured in medium CFM. Immunoblot analysis showed that the level of EGFP-LC3II (45 kDa) was increased in a time-dependent manner in cells grown in CFM.

Techniques Used: Stable Transfection, Transfection, Incubation, Microscopy, Blocking Assay, Cell Culture, MTT Assay, Activation Assay

24) Product Images from "Maturation of dendritic cells depends on proteolytic cleavage by cathepsin X"

Article Title: Maturation of dendritic cells depends on proteolytic cleavage by cathepsin X

Journal: Journal of Leukocyte Biology

doi: 10.1189/jlb.0508285

Cathepsin X enables podosome formation. Immature DCs on Day 5 of differentiation were centrifuged with cytospin (Cytofuge) for 6 min at 1000 rpm onto glass cover slides. Actin was labeled with phalloidin-tetramethylrhodamine B isothiocyanate conjugate (500 ng/ml) for 30 min at room temperature. Podosome formation, present in control, immature DCs (A), is prevented by inhibition of cathepsin X during DC differentiation (B). Adhesion of maturing DCs coincides with β 2 -integrin activation and colocalization with actin (C). Immature and mature DCs were labeled by centrifugation with cytospin (Cytofuge), whereas maturing, adherent DCs were labeled by seeding immature DCs onto glass coverslips in 24-well plates in the presence of 20 ng/ml LPS and allowing adherence for 20 h. The active form of β 2 integrin was labeled with mAb 24 (green fluorescence) and colocalized with actin (red fluorescence) in adherent, mature DCs. Meanwhile, formation of typical dendrites in mature DCs (D) was not inhibited by cathepsin X inhibition (E). Original scale bars represent 20 μm. Fluorescence microscopy was performed using a Carl Zeiss LSM 510 confocal microscope. Images were analyzed using Carl Zeiss LSM image software 3.0.
Figure Legend Snippet: Cathepsin X enables podosome formation. Immature DCs on Day 5 of differentiation were centrifuged with cytospin (Cytofuge) for 6 min at 1000 rpm onto glass cover slides. Actin was labeled with phalloidin-tetramethylrhodamine B isothiocyanate conjugate (500 ng/ml) for 30 min at room temperature. Podosome formation, present in control, immature DCs (A), is prevented by inhibition of cathepsin X during DC differentiation (B). Adhesion of maturing DCs coincides with β 2 -integrin activation and colocalization with actin (C). Immature and mature DCs were labeled by centrifugation with cytospin (Cytofuge), whereas maturing, adherent DCs were labeled by seeding immature DCs onto glass coverslips in 24-well plates in the presence of 20 ng/ml LPS and allowing adherence for 20 h. The active form of β 2 integrin was labeled with mAb 24 (green fluorescence) and colocalized with actin (red fluorescence) in adherent, mature DCs. Meanwhile, formation of typical dendrites in mature DCs (D) was not inhibited by cathepsin X inhibition (E). Original scale bars represent 20 μm. Fluorescence microscopy was performed using a Carl Zeiss LSM 510 confocal microscope. Images were analyzed using Carl Zeiss LSM image software 3.0.

Techniques Used: Labeling, Inhibition, Activation Assay, Centrifugation, Fluorescence, Microscopy, Software

Phenotypic characteristics of DC maturation. Surface marker expression was determined by FACS analysis of mature DCs stimulated for 48 h with LPS (20 ng/ml; A). Broken line shows staining with an isotype control, dotted line the staining of mature DCs, and solid line staining of DCs matured in the presence of the cathepsin X inhibitor. The results are representative of three independent experiments, and the average MFI for control and mAb 2F12-treated DCs is given in the right top corner in histograms. Surface expression of cathepsin X (solid line) was evaluated in adherent maturing and floating mature DCs (B and C). Immature DCs were stimulated with 20 ng/ml LPS for 48 h or TNF-α for 5 days and analyzed for cathepsin X (katX) membrane (nonpermeabilized, DC) or intracellular (permeabilized, DC) expression. Broken lines represent isotype controls (B). Confocal images of cathepsin X translocation to the plasma membrane in maturing, adherent DCs (C). Original scale bars represent 20 μm. Fluorescence microscopy was performed using a Carl Zeiss LSM 510 confocal microscope. Images were analyzed using Carl Zeiss LSM image software 3.0.
Figure Legend Snippet: Phenotypic characteristics of DC maturation. Surface marker expression was determined by FACS analysis of mature DCs stimulated for 48 h with LPS (20 ng/ml; A). Broken line shows staining with an isotype control, dotted line the staining of mature DCs, and solid line staining of DCs matured in the presence of the cathepsin X inhibitor. The results are representative of three independent experiments, and the average MFI for control and mAb 2F12-treated DCs is given in the right top corner in histograms. Surface expression of cathepsin X (solid line) was evaluated in adherent maturing and floating mature DCs (B and C). Immature DCs were stimulated with 20 ng/ml LPS for 48 h or TNF-α for 5 days and analyzed for cathepsin X (katX) membrane (nonpermeabilized, DC) or intracellular (permeabilized, DC) expression. Broken lines represent isotype controls (B). Confocal images of cathepsin X translocation to the plasma membrane in maturing, adherent DCs (C). Original scale bars represent 20 μm. Fluorescence microscopy was performed using a Carl Zeiss LSM 510 confocal microscope. Images were analyzed using Carl Zeiss LSM image software 3.0.

Techniques Used: Marker, Expressing, FACS, Staining, Translocation Assay, Fluorescence, Microscopy, Software

Cathepsin X colocalizes with the Mac-1 integrin receptor in maturing, adherent DCs. Cathepsin X was labeled with Alexa Fluor 488-labeled mouse 2F12 mAb that recognizes the mature, active form. Absence of colocalization of cathepsin X (green fluorescence) and Mac-1 integrin receptor (red fluorescence) in immature DC (iDC; A) and mature DCs (C) is shown. Differential interference contrast images are shown, respectively. Original bars, 20 μm. Maturing, adherent DCs were labeled by seeding immature DCs onto glass coverslips in 24-well plates in the presence of 20 ng/ml LPS and allowing adherence for 20 h. The translocation of cathepsin X in maturating, adherent DCs to the plasma membrane, where it colocalizes with the Mac-1 integrin receptor, is demonstrated (B). Original scale bars represent 20 μm. Fluorescence microscopy was performed using a Carl Zeiss LSM 510 confocal microscope. Images were analyzed using Carl Zeiss LSM image software 3.0.
Figure Legend Snippet: Cathepsin X colocalizes with the Mac-1 integrin receptor in maturing, adherent DCs. Cathepsin X was labeled with Alexa Fluor 488-labeled mouse 2F12 mAb that recognizes the mature, active form. Absence of colocalization of cathepsin X (green fluorescence) and Mac-1 integrin receptor (red fluorescence) in immature DC (iDC; A) and mature DCs (C) is shown. Differential interference contrast images are shown, respectively. Original bars, 20 μm. Maturing, adherent DCs were labeled by seeding immature DCs onto glass coverslips in 24-well plates in the presence of 20 ng/ml LPS and allowing adherence for 20 h. The translocation of cathepsin X in maturating, adherent DCs to the plasma membrane, where it colocalizes with the Mac-1 integrin receptor, is demonstrated (B). Original scale bars represent 20 μm. Fluorescence microscopy was performed using a Carl Zeiss LSM 510 confocal microscope. Images were analyzed using Carl Zeiss LSM image software 3.0.

Techniques Used: Labeling, Fluorescence, Translocation Assay, Microscopy, Software

25) Product Images from "Suberoylanilide Hydroxamic Acid Induces Hypersensitivity to Radiation Therapy in Acute Myelogenous Leukemia Cells Expressing Constitutively Active FLT3 Mutants"

Article Title: Suberoylanilide Hydroxamic Acid Induces Hypersensitivity to Radiation Therapy in Acute Myelogenous Leukemia Cells Expressing Constitutively Active FLT3 Mutants

Journal: PLoS ONE

doi: 10.1371/journal.pone.0084515

The effects of constitutively activated FLT3 mutants on end-joining DNA repair are associated with inhibited DNA-PKCs activity. (A) Immunoblot assay. Whole cell extracts from TF1 cells with stable expression of different forms of FLT3 were analyzed by immunoblot assays with anti-DNA LigIII, DNA-PKCs, Ku70, Ku86 and RAD51 antibodies. β-actin was included to confirm equivalent protein loading. (B) Representative images of the changes for nuclear phosphor-DNA-PKCs /γ-H2A.X co-foci in response to IR treatment. Engineered TF1 cells were irradiated with 1.2 Gy. Cells were collected one hour later for immunofluorescence staining with anti-γ-H2A.X (green) and anti-phosphor-DNA-pK (T2609, red). Nuclei were stained with DAPI (blue). Images were acquired with LSM 510 confocal microscope (Zeiss) with 80X objective and processed by Photoshop (Adobe). (C) Diagram shows changes in the fraction of cells with phosphor-DNA-PKCs/γ-H2A.X co-foci in engineered TF1 cells. (D) Expressing FLT3 mutants does not change the inhibitory effects of SAHA on the formation of RAD51/γ-H2A.X nuclear co-foci in irradiated TF1 cells. Engineered TF1 cells were collected 7 hours later after 1.2 Gy of irradiation, and immunofluorescence stained with anti-γ-H2A.X (green) and anti-RAD51 (red). Nuclei were stained with DAPI (blue). Error bars indicate standard deviation. * indicates significance (P
Figure Legend Snippet: The effects of constitutively activated FLT3 mutants on end-joining DNA repair are associated with inhibited DNA-PKCs activity. (A) Immunoblot assay. Whole cell extracts from TF1 cells with stable expression of different forms of FLT3 were analyzed by immunoblot assays with anti-DNA LigIII, DNA-PKCs, Ku70, Ku86 and RAD51 antibodies. β-actin was included to confirm equivalent protein loading. (B) Representative images of the changes for nuclear phosphor-DNA-PKCs /γ-H2A.X co-foci in response to IR treatment. Engineered TF1 cells were irradiated with 1.2 Gy. Cells were collected one hour later for immunofluorescence staining with anti-γ-H2A.X (green) and anti-phosphor-DNA-pK (T2609, red). Nuclei were stained with DAPI (blue). Images were acquired with LSM 510 confocal microscope (Zeiss) with 80X objective and processed by Photoshop (Adobe). (C) Diagram shows changes in the fraction of cells with phosphor-DNA-PKCs/γ-H2A.X co-foci in engineered TF1 cells. (D) Expressing FLT3 mutants does not change the inhibitory effects of SAHA on the formation of RAD51/γ-H2A.X nuclear co-foci in irradiated TF1 cells. Engineered TF1 cells were collected 7 hours later after 1.2 Gy of irradiation, and immunofluorescence stained with anti-γ-H2A.X (green) and anti-RAD51 (red). Nuclei were stained with DAPI (blue). Error bars indicate standard deviation. * indicates significance (P

Techniques Used: Activity Assay, Expressing, Irradiation, Immunofluorescence, Staining, Microscopy, Standard Deviation

26) Product Images from "M-CSF Signals through the MAPK/ERK Pathway via Sp1 to Induce VEGF Production and Induces Angiogenesis In Vivo"

Article Title: M-CSF Signals through the MAPK/ERK Pathway via Sp1 to Induce VEGF Production and Induces Angiogenesis In Vivo

Journal: PLoS ONE

doi: 10.1371/journal.pone.0003405

M-CSF induces Sp1 nuclear localization in an ERK-dependent manner. A) Human monocytes were left non-stimulated (NS) or stimulated with rhM-CSF (100 ng/ml) (M-CSF) for 30 minutes. Nuclear lysates were isolated and normalized for total protein. Sp1 that translocated into the nucleus in response to M-CSF was analyzed using a biotinylated Sp1 DNA sequence bound to a streptavidin-coated plate, a polyclonal rabbit anti-Sp1 primary antibody, a HRP-conjugated goat anti-rabbit IgG secondary antibody, and TMB substrate. The absorbance was read at 450 nm to reflect Sp1 within the nucleus. These data represent the mean±SEM from four independent blood donors. B) Human monocytes were starved for 6 hours, inhibited for 30 minutes with U0126 (10 µM) or DMSO (vehicle control) and left non-stimulated (Non-stimulated) or treated with rhM-CSF for 6 hours (M-CSF+DMSO) and (M-CSF+U0126) . The cells were fixed, permeablized, and stained with a normal IgG control antibody (top row) or a primary antibody targeting Sp1 followed by subsequent staining with Alexa 594-conjugated secondary antibody, targeting Sp1 (red) , and with DAPI stain designating the nucleus (blue) . Images were captured using the Zeiss LSM 510 confocal microscope. These pictures are representative of four individual monocyte donors. C) Quantification of Sp1 localization to the nucleus of monocytes (horseshoe-shaped nuclei) using Image J software. This data represents mean+/−SEM of cells from four individual trials.
Figure Legend Snippet: M-CSF induces Sp1 nuclear localization in an ERK-dependent manner. A) Human monocytes were left non-stimulated (NS) or stimulated with rhM-CSF (100 ng/ml) (M-CSF) for 30 minutes. Nuclear lysates were isolated and normalized for total protein. Sp1 that translocated into the nucleus in response to M-CSF was analyzed using a biotinylated Sp1 DNA sequence bound to a streptavidin-coated plate, a polyclonal rabbit anti-Sp1 primary antibody, a HRP-conjugated goat anti-rabbit IgG secondary antibody, and TMB substrate. The absorbance was read at 450 nm to reflect Sp1 within the nucleus. These data represent the mean±SEM from four independent blood donors. B) Human monocytes were starved for 6 hours, inhibited for 30 minutes with U0126 (10 µM) or DMSO (vehicle control) and left non-stimulated (Non-stimulated) or treated with rhM-CSF for 6 hours (M-CSF+DMSO) and (M-CSF+U0126) . The cells were fixed, permeablized, and stained with a normal IgG control antibody (top row) or a primary antibody targeting Sp1 followed by subsequent staining with Alexa 594-conjugated secondary antibody, targeting Sp1 (red) , and with DAPI stain designating the nucleus (blue) . Images were captured using the Zeiss LSM 510 confocal microscope. These pictures are representative of four individual monocyte donors. C) Quantification of Sp1 localization to the nucleus of monocytes (horseshoe-shaped nuclei) using Image J software. This data represents mean+/−SEM of cells from four individual trials.

Techniques Used: Isolation, Sequencing, Staining, Microscopy, Software

27) Product Images from "The dependency of solute diffusion on molecular weight and shape in intact bone"

Article Title: The dependency of solute diffusion on molecular weight and shape in intact bone

Journal: Bone

doi: 10.1016/j.bone.2009.07.076

Experimental setup for FRAP measurements of tracer diffusion in intact murine tibiae. (A) The FRAP experiment was performed using a Zeiss LSM 510 confocal module (not shown in the picture) attached to an Axioobserver Z1 invert microscope (1). A lens inverter (2) was used to direct the imaging laser beam to an elevated platform (3) beside the microscope, which was consisted of a height-adjustable jack and a xy table. The animal was placed in a water bath (4) perfused with 25°C PBS solution (heating elements not shown). (B) The left tibia was fastened rigidly at the knee using a rod with a cup-shaped end (5) and at the ankle using a slit and a rubber band (6) under a small compression force. The tibial anterior-medial surface (7) was surgically exposed for FRAP imaging.
Figure Legend Snippet: Experimental setup for FRAP measurements of tracer diffusion in intact murine tibiae. (A) The FRAP experiment was performed using a Zeiss LSM 510 confocal module (not shown in the picture) attached to an Axioobserver Z1 invert microscope (1). A lens inverter (2) was used to direct the imaging laser beam to an elevated platform (3) beside the microscope, which was consisted of a height-adjustable jack and a xy table. The animal was placed in a water bath (4) perfused with 25°C PBS solution (heating elements not shown). (B) The left tibia was fastened rigidly at the knee using a rod with a cup-shaped end (5) and at the ankle using a slit and a rubber band (6) under a small compression force. The tibial anterior-medial surface (7) was surgically exposed for FRAP imaging.

Techniques Used: Diffusion-based Assay, Microscopy, Imaging

28) Product Images from "Tensin1 and a previously undocumented family member, tensin2, positively regulate cell migration"

Article Title: Tensin1 and a previously undocumented family member, tensin2, positively regulate cell migration

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.022518699

Localization of GFP–tensin2 fusion proteins in NIH 3T3 cells. Cells grown on coverslips were transfected with GFP or GFP–tensin2 constructs. After 24 h, cells were fixed and labeled for tensin1, vinculin, and actin stress fibers. Cells were then visualized with a Zeiss LSM 510 laser scanning microscope. Arrows indicate focal adhesions. Arrowhead shows the ECM contacts.
Figure Legend Snippet: Localization of GFP–tensin2 fusion proteins in NIH 3T3 cells. Cells grown on coverslips were transfected with GFP or GFP–tensin2 constructs. After 24 h, cells were fixed and labeled for tensin1, vinculin, and actin stress fibers. Cells were then visualized with a Zeiss LSM 510 laser scanning microscope. Arrows indicate focal adhesions. Arrowhead shows the ECM contacts.

Techniques Used: Transfection, Construct, Labeling, Laser-Scanning Microscopy

29) Product Images from "Absence of an N-Linked Glycosylation Motif in the Glycoprotein of the Live-Attenuated Argentine Hemorrhagic Fever Vaccine, Candid #1, Results in Its Improper Processing, and Reduced Surface Expression"

Article Title: Absence of an N-Linked Glycosylation Motif in the Glycoprotein of the Live-Attenuated Argentine Hemorrhagic Fever Vaccine, Candid #1, Results in Its Improper Processing, and Reduced Surface Expression

Journal: Frontiers in Cellular and Infection Microbiology

doi: 10.3389/fcimb.2017.00020

Intracellular localization of JUNV GPC relative to the ER and cell surface . Monolayers of HEK293 cells were transfected with CMV-driven expression plasmids expressing Romero, XJ13, XJ44, Candid, Romero expressing the T168A substitution (RomT168A), and Candid expressing the A168T reversion (CanA168T). At 36 h post-transfection, cells were permeabilized and stained with both an ER-selective fluorescent dye (red) and Hoechst 33342 nuclear dye (blue), and an anti-JUNV GPC primary antibody followed by a conjugated AlexaFluor TM secondary antibody (green). Cells were imaged using a Zeiss LSM 510 confocal microscope. Both the merged images on a single Z plane (left) and a cross-section of a single representative cell sectioned along along the Z axis (right) are included.
Figure Legend Snippet: Intracellular localization of JUNV GPC relative to the ER and cell surface . Monolayers of HEK293 cells were transfected with CMV-driven expression plasmids expressing Romero, XJ13, XJ44, Candid, Romero expressing the T168A substitution (RomT168A), and Candid expressing the A168T reversion (CanA168T). At 36 h post-transfection, cells were permeabilized and stained with both an ER-selective fluorescent dye (red) and Hoechst 33342 nuclear dye (blue), and an anti-JUNV GPC primary antibody followed by a conjugated AlexaFluor TM secondary antibody (green). Cells were imaged using a Zeiss LSM 510 confocal microscope. Both the merged images on a single Z plane (left) and a cross-section of a single representative cell sectioned along along the Z axis (right) are included.

Techniques Used: Gel Permeation Chromatography, Transfection, Expressing, Staining, Microscopy

30) Product Images from "Complement Component C3 and Complement Receptor Type 3 Contribute to the Phagocytosis and Clearance of Fibrillar A? by Microglia"

Article Title: Complement Component C3 and Complement Receptor Type 3 Contribute to the Phagocytosis and Clearance of Fibrillar A? by Microglia

Journal: Glia

doi: 10.1002/glia.22331

Primary wildtype microglia take up microinjected FLfAβ 42 and transport it to lysosomes in vivo . A–C : Five days after the stereotaxic injection of 0.6 µg FLfAβ 42 into the frontal cortex, wildtype C57BL/6 mouse brains were sectioned and incubated with anti-Iba-1 (A), anti-CD68 (B) or anti-LAMP-1 (C) antibodies. After washing with PBS, sections were incubated with appropriate Alexa Fluor second antibodies and nuclei were visualized by TO Pro-3 staining. Images were obtained as a z-series stack using a Zeiss LSM 510 confocal microscope. White scale bar = 50 µm; red scale bar = 10 µm.
Figure Legend Snippet: Primary wildtype microglia take up microinjected FLfAβ 42 and transport it to lysosomes in vivo . A–C : Five days after the stereotaxic injection of 0.6 µg FLfAβ 42 into the frontal cortex, wildtype C57BL/6 mouse brains were sectioned and incubated with anti-Iba-1 (A), anti-CD68 (B) or anti-LAMP-1 (C) antibodies. After washing with PBS, sections were incubated with appropriate Alexa Fluor second antibodies and nuclei were visualized by TO Pro-3 staining. Images were obtained as a z-series stack using a Zeiss LSM 510 confocal microscope. White scale bar = 50 µm; red scale bar = 10 µm.

Techniques Used: In Vivo, Injection, Incubation, Staining, Microscopy

31) Product Images from "Characterization of the Expression, Intracellular Localization, and Replication Complex Association of the Putative Mouse Hepatitis Virus RNA-Dependent RNA Polymerase"

Article Title: Characterization of the Expression, Intracellular Localization, and Replication Complex Association of the Putative Mouse Hepatitis Virus RNA-Dependent RNA Polymerase

Journal: Journal of Virology

doi: 10.1128/JVI.77.19.10515-10527.2003

Time course of Pol localization during MHV infection. MHV-infected DBT cells were fixed at the indicated times p.i. prior to preparation for indirect immunofluorescence microscopy as described in Materials and Methods. Cells were imaged on a Zeiss LSM 510 confocal microscope. Images are single confocal slices taken by using a 40× objective and are representative of the cell population. (A) Dual-label imaging of Pol (green) and N (red) at 5.5 h p.i. or of Pol(green) and M (red) at 5.5 and 9 h p.i. (B) Dual-label imaging of p65 (green) and N (red) at 5.5 h p.i. or of p65 (green) and M (red) at 5.5 and 9 h p.i. (C) Dual-label imaging of Hel (green) and N (red) at 5.5 h p.i. or of Hel (green) and M (red) at 5.5 and 9 h p.i. Merged images are shown with areas of colocalization in yellow.
Figure Legend Snippet: Time course of Pol localization during MHV infection. MHV-infected DBT cells were fixed at the indicated times p.i. prior to preparation for indirect immunofluorescence microscopy as described in Materials and Methods. Cells were imaged on a Zeiss LSM 510 confocal microscope. Images are single confocal slices taken by using a 40× objective and are representative of the cell population. (A) Dual-label imaging of Pol (green) and N (red) at 5.5 h p.i. or of Pol(green) and M (red) at 5.5 and 9 h p.i. (B) Dual-label imaging of p65 (green) and N (red) at 5.5 h p.i. or of p65 (green) and M (red) at 5.5 and 9 h p.i. (C) Dual-label imaging of Hel (green) and N (red) at 5.5 h p.i. or of Hel (green) and M (red) at 5.5 and 9 h p.i. Merged images are shown with areas of colocalization in yellow.

Techniques Used: Infection, Immunofluorescence, Microscopy, Imaging

32) Product Images from "Protein arginine N-methyltransferase 2 reverses tamoxifen resistance in breast cancer cells through suppression of ER-α36"

Article Title: Protein arginine N-methyltransferase 2 reverses tamoxifen resistance in breast cancer cells through suppression of ER-α36

Journal: Oncology Reports

doi: 10.3892/or.2018.6350

Interaction of PRMT2 and ER-α36. (A) Subcellular distribution of PRMT2 and ER-α36 in MCF-7 and MDA-MB-231 cells. Cells were transiently co-transfected with N-terminal GFP-tagged PRMT2 and pcDNA3.1-Myc-His-ER-α36 for 48 h, and then cells were viewed under a Zeiss LSM 510 confocal microscope. Cells transiently transfected with the pcDNA3.1-Myc-His vector were used as control. Green represents the pixel intensity distribution of the GFP signal, blue depicts the profile of the exclusively nuclear DAPI staining, and localization of ER-α36 (in red) was determined by Cy3-conjugated antibody. (B) Interaction of PRMT2 with ER-α36 in vitro . GST, GST-ER-α36 and GST-ER-α66 fusion proteins immobilized on beads were mixed with recombinant human His-tag-PRMT2 protein. Bound proteins were subjected to SDS/PAGE separation, followed by immunoblotting. (C) Interaction between PRMT2 and ER-α36 in vivo . MCF-7 and MDA-MB-231 cells were co-transfected with the expression vectors for PRMT2 and ER-α36 as indicated. Lysates from the transfected cells were immunoprecipitated (IP) using PRMT2 antibody and the immunoprecipitates were probed with an ER-α36 antibody or in reverse. PRMT2, protein arginine N-methyltransferase 2.
Figure Legend Snippet: Interaction of PRMT2 and ER-α36. (A) Subcellular distribution of PRMT2 and ER-α36 in MCF-7 and MDA-MB-231 cells. Cells were transiently co-transfected with N-terminal GFP-tagged PRMT2 and pcDNA3.1-Myc-His-ER-α36 for 48 h, and then cells were viewed under a Zeiss LSM 510 confocal microscope. Cells transiently transfected with the pcDNA3.1-Myc-His vector were used as control. Green represents the pixel intensity distribution of the GFP signal, blue depicts the profile of the exclusively nuclear DAPI staining, and localization of ER-α36 (in red) was determined by Cy3-conjugated antibody. (B) Interaction of PRMT2 with ER-α36 in vitro . GST, GST-ER-α36 and GST-ER-α66 fusion proteins immobilized on beads were mixed with recombinant human His-tag-PRMT2 protein. Bound proteins were subjected to SDS/PAGE separation, followed by immunoblotting. (C) Interaction between PRMT2 and ER-α36 in vivo . MCF-7 and MDA-MB-231 cells were co-transfected with the expression vectors for PRMT2 and ER-α36 as indicated. Lysates from the transfected cells were immunoprecipitated (IP) using PRMT2 antibody and the immunoprecipitates were probed with an ER-α36 antibody or in reverse. PRMT2, protein arginine N-methyltransferase 2.

Techniques Used: Multiple Displacement Amplification, Transfection, Microscopy, Plasmid Preparation, Staining, In Vitro, Recombinant, SDS Page, In Vivo, Expressing, Immunoprecipitation

33) Product Images from "Identification and Functional Characterization of Novel Mutations in the Melanocortin-4 Receptor"

Article Title: Identification and Functional Characterization of Novel Mutations in the Melanocortin-4 Receptor

Journal: Obesity Facts

doi: 10.1159/000321565

Localization of wild-type and mutant MC4R. Images were made with a Zeiss LSM 510 confocal microscope equipped with argon (excitation, 488 nm) and helium-neon (excitation, 543 and 633 nm) lasers. The first panel shows EGFP fluorescence (excitation 488 nm). The second panel visualizes fluorescence of the tetramethyl-rhodamine wheat-germ agglutinin conjugate (TMR-WGA) which binds to the plasma membrane (PM) (excitation 543 nm). A merged view is shown in the third panel. A Empty pEGFP-N1 expression vector: EGFP is expressed ubiquitously throughout the cell, without co-localization with the PM B MC4R wild type: co-localization of EGFP and TMR-WGA fluorescence indicates the localization of the wild-type MC4R on the PM. C MC4R I186V: co-localization of green and red fluorescence indicates that this mutant receptor is present on the PM. D MC4R P260Q: no co-localization of EGFP and TMR-WGA is present on the merged view, indicating that this mutant MC4R is not expressed on the PM. E MC4R F280L: the mutation causes intracellular retention of the receptor since no colocalization of EGFP and TMR-WGA is seen.
Figure Legend Snippet: Localization of wild-type and mutant MC4R. Images were made with a Zeiss LSM 510 confocal microscope equipped with argon (excitation, 488 nm) and helium-neon (excitation, 543 and 633 nm) lasers. The first panel shows EGFP fluorescence (excitation 488 nm). The second panel visualizes fluorescence of the tetramethyl-rhodamine wheat-germ agglutinin conjugate (TMR-WGA) which binds to the plasma membrane (PM) (excitation 543 nm). A merged view is shown in the third panel. A Empty pEGFP-N1 expression vector: EGFP is expressed ubiquitously throughout the cell, without co-localization with the PM B MC4R wild type: co-localization of EGFP and TMR-WGA fluorescence indicates the localization of the wild-type MC4R on the PM. C MC4R I186V: co-localization of green and red fluorescence indicates that this mutant receptor is present on the PM. D MC4R P260Q: no co-localization of EGFP and TMR-WGA is present on the merged view, indicating that this mutant MC4R is not expressed on the PM. E MC4R F280L: the mutation causes intracellular retention of the receptor since no colocalization of EGFP and TMR-WGA is seen.

Techniques Used: Mutagenesis, Microscopy, Fluorescence, Whole Genome Amplification, Expressing, Plasmid Preparation

34) Product Images from "Complement Component C3 and Complement Receptor Type 3 Contribute to the Phagocytosis and Clearance of Fibrillar A? by Microglia"

Article Title: Complement Component C3 and Complement Receptor Type 3 Contribute to the Phagocytosis and Clearance of Fibrillar A? by Microglia

Journal: Glia

doi: 10.1002/glia.22331

Primary wildtype microglia take up microinjected FLfAβ 42 and transport it to lysosomes in vivo . A–C : Five days after the stereotaxic injection of 0.6 µg FLfAβ 42 into the frontal cortex, wildtype C57BL/6 mouse brains were sectioned and incubated with anti-Iba-1 (A), anti-CD68 (B) or anti-LAMP-1 (C) antibodies. After washing with PBS, sections were incubated with appropriate Alexa Fluor second antibodies and nuclei were visualized by TO Pro-3 staining. Images were obtained as a z-series stack using a Zeiss LSM 510 confocal microscope. White scale bar = 50 µm; red scale bar = 10 µm.
Figure Legend Snippet: Primary wildtype microglia take up microinjected FLfAβ 42 and transport it to lysosomes in vivo . A–C : Five days after the stereotaxic injection of 0.6 µg FLfAβ 42 into the frontal cortex, wildtype C57BL/6 mouse brains were sectioned and incubated with anti-Iba-1 (A), anti-CD68 (B) or anti-LAMP-1 (C) antibodies. After washing with PBS, sections were incubated with appropriate Alexa Fluor second antibodies and nuclei were visualized by TO Pro-3 staining. Images were obtained as a z-series stack using a Zeiss LSM 510 confocal microscope. White scale bar = 50 µm; red scale bar = 10 µm.

Techniques Used: In Vivo, Injection, Incubation, Staining, Microscopy

35) Product Images from "A Novel Fluorescence Resonance Energy Transfer Assay Demonstrates that the Human Immunodeficiency Virus Type 1 Pr55Gag I Domain Mediates Gag-Gag Interactions"

Article Title: A Novel Fluorescence Resonance Energy Transfer Assay Demonstrates that the Human Immunodeficiency Virus Type 1 Pr55Gag I Domain Mediates Gag-Gag Interactions

Journal: Journal of Virology

doi: 10.1128/JVI.78.3.1230-1242.2004

Visualization of Gag-Gag FRET by confocal microscopy. Gag-CFP and Gag-YFP were cotransfected in Mel JuSo cells, and images were obtained with a Zeiss LSM 510 confocal microscope equipped with a Meta multichannel detector. Fluorescence excitation was carried out at 458 nm; emission images prepared by linear unmixing techniques for CFP (left panels) and YFP (right panels) are shown. (A) CFP emission image demonstrating weak membrane fluorescence in the indicated region before photobleaching. (B) FRET image indicating efficient plasma membrane Gag-Gag FRET prior to photobleaching of YFP. (C) CFP emission image following photobleaching of YFP in the circled region. Note the increase in CFP emission following YFP photobleaching within the indicated region. (D) FRET image following photobleaching of the indicated area of the cell.
Figure Legend Snippet: Visualization of Gag-Gag FRET by confocal microscopy. Gag-CFP and Gag-YFP were cotransfected in Mel JuSo cells, and images were obtained with a Zeiss LSM 510 confocal microscope equipped with a Meta multichannel detector. Fluorescence excitation was carried out at 458 nm; emission images prepared by linear unmixing techniques for CFP (left panels) and YFP (right panels) are shown. (A) CFP emission image demonstrating weak membrane fluorescence in the indicated region before photobleaching. (B) FRET image indicating efficient plasma membrane Gag-Gag FRET prior to photobleaching of YFP. (C) CFP emission image following photobleaching of YFP in the circled region. Note the increase in CFP emission following YFP photobleaching within the indicated region. (D) FRET image following photobleaching of the indicated area of the cell.

Techniques Used: Confocal Microscopy, Microscopy, Fluorescence

Analysis of Gag-Gag interactions by FRET microscopy and spectral analysis. (A) Gag-CFP and Gag-YFP were cotransfected in Mel JuSo cells, and images were obtained with a Zeiss LSM 510-Meta confocal microscope. The image represents YFP excitation-emission before photobleaching. The arrow indicates the selected plasma membrane region to be bleached. (B) The same cell as that shown in panel A is depicted following photobleaching at 514 nm. (C) Emission scans were obtained from the selected region of interest, with excitation at 458 nm (CFP excitation), before (solid line) and after (dashed line) photobleaching of YFP. Peak emission wavelengths for CFP (secondary peak) and YFP are indicated. (D) Gag-YFP distribution before bleaching. (E) Gag-YFP after photobleaching of indicated region. (F) Spectra obtained before (solid line) and after (dashed line) photobleaching of cells from panels D and E. (G) Cotransfection of Myr −  Gag-YFP and Myr −  Gag-CFP before photobleaching. (H) The same cell as that shown in panel G is depicted after photobleaching of indicated region of the cytoplasm. (I) Spectral analysis of the cell shown in panels G and H.
Figure Legend Snippet: Analysis of Gag-Gag interactions by FRET microscopy and spectral analysis. (A) Gag-CFP and Gag-YFP were cotransfected in Mel JuSo cells, and images were obtained with a Zeiss LSM 510-Meta confocal microscope. The image represents YFP excitation-emission before photobleaching. The arrow indicates the selected plasma membrane region to be bleached. (B) The same cell as that shown in panel A is depicted following photobleaching at 514 nm. (C) Emission scans were obtained from the selected region of interest, with excitation at 458 nm (CFP excitation), before (solid line) and after (dashed line) photobleaching of YFP. Peak emission wavelengths for CFP (secondary peak) and YFP are indicated. (D) Gag-YFP distribution before bleaching. (E) Gag-YFP after photobleaching of indicated region. (F) Spectra obtained before (solid line) and after (dashed line) photobleaching of cells from panels D and E. (G) Cotransfection of Myr − Gag-YFP and Myr − Gag-CFP before photobleaching. (H) The same cell as that shown in panel G is depicted after photobleaching of indicated region of the cytoplasm. (I) Spectral analysis of the cell shown in panels G and H.

Techniques Used: Microscopy, Cotransfection

The I domain mediates Gag-Gag interactions. (A) Gag fusion constructs were expressed in 293T cells and harvested by Dounce homogenization. Whole-cell lysates were analyzed by fluorometry. The intensities of curves were normalized based on the amount of YFP expressed. The YFP-alone emission curve for each pair of constructs was subtracted from the curve of the CFP-YFP combination (i.e., MA-CFP-MA-YFP curve − MA-YFP-alone curve), resulting in an emission peak at 527 nm representing FRET with the background subtracted for each construct. (B) Gag384-CFP and Gag384-YFP were cotransfected in Mel JuSo cells, and images were obtained with a Zeiss LSM 510-Meta confocal microscope. The image represents YFP excitation-emission before photobleaching. (C) The same cell as that shown in panel B following photobleaching of the indicated regions at 514 nm. (D) Emission spectra were obtained from the selected regions of interest before (diamonds) and after (squares) photobleaching. The excitation wavelength for the scan was 458 nm. (E) Gag384(R380,384A)-CFP cotransfected with Gag384(R380,384A)-YFP. The image represents YFP excitation-emission. (F) The same cell as that shown in panel E following photobleaching at 514 nm. (G) Spectral data obtained from the indicated regions of panels E and F before bleaching (diamonds) and after bleaching (squares).
Figure Legend Snippet: The I domain mediates Gag-Gag interactions. (A) Gag fusion constructs were expressed in 293T cells and harvested by Dounce homogenization. Whole-cell lysates were analyzed by fluorometry. The intensities of curves were normalized based on the amount of YFP expressed. The YFP-alone emission curve for each pair of constructs was subtracted from the curve of the CFP-YFP combination (i.e., MA-CFP-MA-YFP curve − MA-YFP-alone curve), resulting in an emission peak at 527 nm representing FRET with the background subtracted for each construct. (B) Gag384-CFP and Gag384-YFP were cotransfected in Mel JuSo cells, and images were obtained with a Zeiss LSM 510-Meta confocal microscope. The image represents YFP excitation-emission before photobleaching. (C) The same cell as that shown in panel B following photobleaching of the indicated regions at 514 nm. (D) Emission spectra were obtained from the selected regions of interest before (diamonds) and after (squares) photobleaching. The excitation wavelength for the scan was 458 nm. (E) Gag384(R380,384A)-CFP cotransfected with Gag384(R380,384A)-YFP. The image represents YFP excitation-emission. (F) The same cell as that shown in panel E following photobleaching at 514 nm. (G) Spectral data obtained from the indicated regions of panels E and F before bleaching (diamonds) and after bleaching (squares).

Techniques Used: Construct, Homogenization, Microscopy

36) Product Images from "The Adaptor Protein SH2B3 (Lnk) Negatively Regulates Neurite Outgrowth of PC12 Cells and Cortical Neurons"

Article Title: The Adaptor Protein SH2B3 (Lnk) Negatively Regulates Neurite Outgrowth of PC12 Cells and Cortical Neurons

Journal: PLoS ONE

doi: 10.1371/journal.pone.0026433

Expression of SH2B3 in the E18 rat brain cortex and in PC12 cells. (A) Brain slices of E18 rats were subjected to immunofluorescence staining using anti-SH2B3 antibody followed by Alexa Fluor 555-conjugated secondary antibody (shown in red). Enlarged panels from the square box area are shown in the middle column. Secondary antibody controls (without primary antibody) are shown in the panels on the right column. DAPI staining in blue shows the localization of the nucleus. Images were taken using Zeiss LSM 510. Scale bar: 50 µm. (B) Microarray results generated from rat affymetrix chips. Relative mRNA expressions of SH2B3 in PC12-GFP cells treated without (Ctrl) or with 100 ng/ml NGF for 6 h are shown. (C) Relative mRNA expressions of SH2B3 in PC12 cells treated with NGF 50 ng/ml for the indicated time periods were analyzed using Q-PCR. The relative SH2B3 levels were normalized to the levels of GADPH. *: P
Figure Legend Snippet: Expression of SH2B3 in the E18 rat brain cortex and in PC12 cells. (A) Brain slices of E18 rats were subjected to immunofluorescence staining using anti-SH2B3 antibody followed by Alexa Fluor 555-conjugated secondary antibody (shown in red). Enlarged panels from the square box area are shown in the middle column. Secondary antibody controls (without primary antibody) are shown in the panels on the right column. DAPI staining in blue shows the localization of the nucleus. Images were taken using Zeiss LSM 510. Scale bar: 50 µm. (B) Microarray results generated from rat affymetrix chips. Relative mRNA expressions of SH2B3 in PC12-GFP cells treated without (Ctrl) or with 100 ng/ml NGF for 6 h are shown. (C) Relative mRNA expressions of SH2B3 in PC12 cells treated with NGF 50 ng/ml for the indicated time periods were analyzed using Q-PCR. The relative SH2B3 levels were normalized to the levels of GADPH. *: P

Techniques Used: Expressing, Immunofluorescence, Staining, Microarray, Generated, Polymerase Chain Reaction

37) Product Images from "Ubiquitin C-Terminal Hydrolase L1 Is Expressed in Mouse Pituitary Gonadotropes In Vivo and Gonadotrope Cell LinesIn Vitro"

Article Title: Ubiquitin C-Terminal Hydrolase L1 Is Expressed in Mouse Pituitary Gonadotropes In Vivo and Gonadotrope Cell LinesIn Vitro

Journal: Experimental Animals

doi: 10.1538/expanim.63.247

The localization of UCH-L1 protein in αT3-1 and LβT-2 cells. To examine the localization of UCH-L1 protein in αT3-1 (upper panels) and LβT-2 cells (lower panels), immunofluorescent staining of UCH-L1 was conducted. TO-PRO-3 was used to visualize the nuclei (a, e). UCH-L1 (b, f), the merged (c, g) and transparent images (d, h) are presented. Images were photographed using a Zeiss LSM 510 confocal microscope.
Figure Legend Snippet: The localization of UCH-L1 protein in αT3-1 and LβT-2 cells. To examine the localization of UCH-L1 protein in αT3-1 (upper panels) and LβT-2 cells (lower panels), immunofluorescent staining of UCH-L1 was conducted. TO-PRO-3 was used to visualize the nuclei (a, e). UCH-L1 (b, f), the merged (c, g) and transparent images (d, h) are presented. Images were photographed using a Zeiss LSM 510 confocal microscope.

Techniques Used: Staining, Microscopy

38) Product Images from "A modular chitin-binding protease associated with hemocytes and hemolymph in the mosquito Anopheles gambiae"

Article Title: A modular chitin-binding protease associated with hemocytes and hemolymph in the mosquito Anopheles gambiae

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi:

Immunolocalization of Sp22D protein in adult tissues and cell lines. Nuclei are counterstained with anti-histone mAb (red in A and B , blue in F – H ). ( A ) Sp22D staining (green) in a perfused hemocyte from a female mosquito. ( B ) Two distinct hemocyte-like subpopulations of cell line 3A expressing Sp22D (green) and defensin (blue). ( C – E ) Sections of paraffin-embedded adult mosquitoes showing Sp22D (red) ( C ) in hemocytes attached to muscle, ( D ) in a single hemocyte attached to a trachea, ( E ) in minute tracheal associated cells but not in tracheal epithelial cells (ec) or in fat body (fb). ( F – H ) Whole-mount triple staining of dissected abdomens showing two characteristic rows of binucleated pericardial cells. Arrows highlight pairs of nuclei, counterstained in blue; Sp22D stains green and defensin red. ( F ) Control treated with Sp22D and defensin preimmune sera. ( G ) An unchallenged mosquito showing constitutive presence of Sp22D and absence of defensin. ( H ) Accumulation of defensin as well as Sp22D in a bacterially challenged mosquito; defensin but not Sp22D is also detected in fat body cells (fb, arrowhead; note the unstained large lipid vacuole characteristic of this tissue). In B – E , the fluorescence channel and Nomarski optics of a Zeiss LSM 510 confocal microscope were combined. (Scale bars = 10 μm.)
Figure Legend Snippet: Immunolocalization of Sp22D protein in adult tissues and cell lines. Nuclei are counterstained with anti-histone mAb (red in A and B , blue in F – H ). ( A ) Sp22D staining (green) in a perfused hemocyte from a female mosquito. ( B ) Two distinct hemocyte-like subpopulations of cell line 3A expressing Sp22D (green) and defensin (blue). ( C – E ) Sections of paraffin-embedded adult mosquitoes showing Sp22D (red) ( C ) in hemocytes attached to muscle, ( D ) in a single hemocyte attached to a trachea, ( E ) in minute tracheal associated cells but not in tracheal epithelial cells (ec) or in fat body (fb). ( F – H ) Whole-mount triple staining of dissected abdomens showing two characteristic rows of binucleated pericardial cells. Arrows highlight pairs of nuclei, counterstained in blue; Sp22D stains green and defensin red. ( F ) Control treated with Sp22D and defensin preimmune sera. ( G ) An unchallenged mosquito showing constitutive presence of Sp22D and absence of defensin. ( H ) Accumulation of defensin as well as Sp22D in a bacterially challenged mosquito; defensin but not Sp22D is also detected in fat body cells (fb, arrowhead; note the unstained large lipid vacuole characteristic of this tissue). In B – E , the fluorescence channel and Nomarski optics of a Zeiss LSM 510 confocal microscope were combined. (Scale bars = 10 μm.)

Techniques Used: Staining, Expressing, Fluorescence, Microscopy

39) Product Images from "Affibody-DyLight Conjugates for In Vivo Assessment of HER2 Expression by Near-Infrared Optical Imaging"

Article Title: Affibody-DyLight Conjugates for In Vivo Assessment of HER2 Expression by Near-Infrared Optical Imaging

Journal: PLoS ONE

doi: 10.1371/journal.pone.0041016

Confocal microscopy of HER2-positive (upper panel) and HER2-negative (lower panel) cells exposed to ZHER2-DyLight-488. SK-BR-3 and MDA-MB-468 cells were plated at 2×10 4 cells/chamber and exposed to Z HER2 -DyLight-488 at 0.5 µg/ml. After a 30 min loading step, cells were rinsed and incubation was continued for indicated period of time at 37°C. A half hour before imaging, the nuclei were counterstained with Hoechst 33342 at 0.2 µg/ml. Images were acquired using Zeiss LSM 510 microscope.
Figure Legend Snippet: Confocal microscopy of HER2-positive (upper panel) and HER2-negative (lower panel) cells exposed to ZHER2-DyLight-488. SK-BR-3 and MDA-MB-468 cells were plated at 2×10 4 cells/chamber and exposed to Z HER2 -DyLight-488 at 0.5 µg/ml. After a 30 min loading step, cells were rinsed and incubation was continued for indicated period of time at 37°C. A half hour before imaging, the nuclei were counterstained with Hoechst 33342 at 0.2 µg/ml. Images were acquired using Zeiss LSM 510 microscope.

Techniques Used: Confocal Microscopy, Multiple Displacement Amplification, Incubation, Imaging, Microscopy

40) Product Images from "Histone deacetylase 4 associates with extracellular signal-regulated kinases 1 and 2, and its cellular localization is regulated by oncogenic Ras"

Article Title: Histone deacetylase 4 associates with extracellular signal-regulated kinases 1 and 2, and its cellular localization is regulated by oncogenic Ras

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi:

H-Ras V12 increased the percentage of cells that express HDAC4 in the nucleus. C2C12 cells were transfected with expression vectors containing EGFP, EGFP plus H-Ras V12 , E-HDAC4, or E-HDAC4 plus H-Ras V12 , as indicated. The localization of EGFP and E-HDAC4 was examined by using a confocal microscope (Zeiss) LSM 510 at ×63. GFP, green fluorescent protein; T. Red, Texas Red detection of H-Ras V12 ; Hoechst, Hoechst 33342 detection of DNA (Blue); Merge, overlay of the three other panels.
Figure Legend Snippet: H-Ras V12 increased the percentage of cells that express HDAC4 in the nucleus. C2C12 cells were transfected with expression vectors containing EGFP, EGFP plus H-Ras V12 , E-HDAC4, or E-HDAC4 plus H-Ras V12 , as indicated. The localization of EGFP and E-HDAC4 was examined by using a confocal microscope (Zeiss) LSM 510 at ×63. GFP, green fluorescent protein; T. Red, Texas Red detection of H-Ras V12 ; Hoechst, Hoechst 33342 detection of DNA (Blue); Merge, overlay of the three other panels.

Techniques Used: Transfection, Expressing, Microscopy

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    Carl Zeiss confocal zeiss lsm 510 fluorescence microscope
    Expression of IBV S protein in DF-1 cells infected with the rNDV-vectored IBV vaccine candidates. A Western blot analysis of IBV S protein. DF-1 cell lysates were analyzed 48 hpi by Western blot analysis using polyclonal IBV antisera (top panel). Analysis of incorporation of IBV S protein into purified rNDV virions from infective allantoic fluid by Western blot analysis using polyclonal IBV antisera (bottom panel). M, Marker; 1, rLaSota/wt.S; 2, rLaSota/S(Y1145A) + Fct 12 ; 3, rLaSota/SΔct + Fct 12 ; 4, rLaSota; 5, mock-infected. B Immunofluorescence analysis of the intracellular and surface expression of IBV S protein in DF-1 cells infected with the rNDV-vectored IBV vaccine candidates. The cells were probed with polyclonal IBV antisera followed by detection with FITC-conjugated goat anti-chicken IgG antibodies (green) and DAPI (blue) and subsequently visualized using a confocal <t>Zeiss</t> <t>LSM</t> 510 fluorescence microscope.
    Confocal Zeiss Lsm 510 Fluorescence Microscope, supplied by Carl Zeiss, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/confocal zeiss lsm 510 fluorescence microscope/product/Carl Zeiss
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    Carl Zeiss lsm 510 confocal microscope
    Heterozygosity for Cldn14 increases tumour blood vessel leakage and decreases intratumoural hypoxia. Wild-type, Cldn14-heterozygous and Cldn14-null mice were injected subcutaneously in the flank with 0.5×10 6 B16F10 melanoma or Lewis Lung Carcinoma (LLC) cells. ( A ) At 10 days post inoculation, PE-conjugated anti-PECAM antibody and Hoechst dye were injected via the tail vein prior to sacrifice. Midline sections (100 µm) of snap-frozen tumours were fixed, mounted and imaged using a Zeiss <t>LSM</t> 510 confocal microscope. The extent of Hoechst leakage was measured in z-stacks using ImageJ. Bars show mean Hoechst leakage relative to PECAM signal ± SEM. Blood vessel leakage is increased significantly in Cldn14-het mice when compared with WT and Cldn14-null mice. ( B ) Representative images of Hoechst (blue) and PECAM (red) detection. ( C ) Tumour-bearing mice from each genotype were injected with pimonidazole prior to sacrifice to measure hypoxic areas within the tumour. 8 µm tumour cryosections were then double stained with anti-pimonidazole antibody (green) to highlight hypoxic areas and anti-PECAM antibody to identify blood vessels. The hypoxic index was quantified relative to PECAM staining using image J software. Bars represent mean relative hypoxic index ± SEM. ( D ) Representative images of pimonidazole detection and PECAM-positive blood vessels in tumour sections. Arrows , blood vessels; Asterisks , pimonidazole-positive staining. Scale bars: A 50 µm; D 200 µm. N = 4 tumours per genotype. NSD: not statistically different, * P
    Lsm 510 Confocal Microscope, supplied by Carl Zeiss, used in various techniques. Bioz Stars score: 96/100, based on 4644 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 96 stars, based on 4644 article reviews
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    Expression of IBV S protein in DF-1 cells infected with the rNDV-vectored IBV vaccine candidates. A Western blot analysis of IBV S protein. DF-1 cell lysates were analyzed 48 hpi by Western blot analysis using polyclonal IBV antisera (top panel). Analysis of incorporation of IBV S protein into purified rNDV virions from infective allantoic fluid by Western blot analysis using polyclonal IBV antisera (bottom panel). M, Marker; 1, rLaSota/wt.S; 2, rLaSota/S(Y1145A) + Fct 12 ; 3, rLaSota/SΔct + Fct 12 ; 4, rLaSota; 5, mock-infected. B Immunofluorescence analysis of the intracellular and surface expression of IBV S protein in DF-1 cells infected with the rNDV-vectored IBV vaccine candidates. The cells were probed with polyclonal IBV antisera followed by detection with FITC-conjugated goat anti-chicken IgG antibodies (green) and DAPI (blue) and subsequently visualized using a confocal Zeiss LSM 510 fluorescence microscope.

    Journal: Veterinary Research

    Article Title: Development of a recombinant Newcastle disease virus-vectored vaccine for infectious bronchitis virus variant strains circulating in Egypt

    doi: 10.1186/s13567-019-0631-5

    Figure Lengend Snippet: Expression of IBV S protein in DF-1 cells infected with the rNDV-vectored IBV vaccine candidates. A Western blot analysis of IBV S protein. DF-1 cell lysates were analyzed 48 hpi by Western blot analysis using polyclonal IBV antisera (top panel). Analysis of incorporation of IBV S protein into purified rNDV virions from infective allantoic fluid by Western blot analysis using polyclonal IBV antisera (bottom panel). M, Marker; 1, rLaSota/wt.S; 2, rLaSota/S(Y1145A) + Fct 12 ; 3, rLaSota/SΔct + Fct 12 ; 4, rLaSota; 5, mock-infected. B Immunofluorescence analysis of the intracellular and surface expression of IBV S protein in DF-1 cells infected with the rNDV-vectored IBV vaccine candidates. The cells were probed with polyclonal IBV antisera followed by detection with FITC-conjugated goat anti-chicken IgG antibodies (green) and DAPI (blue) and subsequently visualized using a confocal Zeiss LSM 510 fluorescence microscope.

    Article Snippet: Cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) and cells were visualized under the Confocal Zeiss LSM 510 fluorescence microscope.

    Techniques: Expressing, Infection, Western Blot, Purification, Marker, Immunofluorescence, Fluorescence, Microscopy

    Heterozygosity for Cldn14 increases tumour blood vessel leakage and decreases intratumoural hypoxia. Wild-type, Cldn14-heterozygous and Cldn14-null mice were injected subcutaneously in the flank with 0.5×10 6 B16F10 melanoma or Lewis Lung Carcinoma (LLC) cells. ( A ) At 10 days post inoculation, PE-conjugated anti-PECAM antibody and Hoechst dye were injected via the tail vein prior to sacrifice. Midline sections (100 µm) of snap-frozen tumours were fixed, mounted and imaged using a Zeiss LSM 510 confocal microscope. The extent of Hoechst leakage was measured in z-stacks using ImageJ. Bars show mean Hoechst leakage relative to PECAM signal ± SEM. Blood vessel leakage is increased significantly in Cldn14-het mice when compared with WT and Cldn14-null mice. ( B ) Representative images of Hoechst (blue) and PECAM (red) detection. ( C ) Tumour-bearing mice from each genotype were injected with pimonidazole prior to sacrifice to measure hypoxic areas within the tumour. 8 µm tumour cryosections were then double stained with anti-pimonidazole antibody (green) to highlight hypoxic areas and anti-PECAM antibody to identify blood vessels. The hypoxic index was quantified relative to PECAM staining using image J software. Bars represent mean relative hypoxic index ± SEM. ( D ) Representative images of pimonidazole detection and PECAM-positive blood vessels in tumour sections. Arrows , blood vessels; Asterisks , pimonidazole-positive staining. Scale bars: A 50 µm; D 200 µm. N = 4 tumours per genotype. NSD: not statistically different, * P

    Journal: PLoS ONE

    Article Title: Stromal Claudin14-Heterozygosity, but Not Deletion, Increases Tumour Blood Leakage without Affecting Tumour Growth

    doi: 10.1371/journal.pone.0062516

    Figure Lengend Snippet: Heterozygosity for Cldn14 increases tumour blood vessel leakage and decreases intratumoural hypoxia. Wild-type, Cldn14-heterozygous and Cldn14-null mice were injected subcutaneously in the flank with 0.5×10 6 B16F10 melanoma or Lewis Lung Carcinoma (LLC) cells. ( A ) At 10 days post inoculation, PE-conjugated anti-PECAM antibody and Hoechst dye were injected via the tail vein prior to sacrifice. Midline sections (100 µm) of snap-frozen tumours were fixed, mounted and imaged using a Zeiss LSM 510 confocal microscope. The extent of Hoechst leakage was measured in z-stacks using ImageJ. Bars show mean Hoechst leakage relative to PECAM signal ± SEM. Blood vessel leakage is increased significantly in Cldn14-het mice when compared with WT and Cldn14-null mice. ( B ) Representative images of Hoechst (blue) and PECAM (red) detection. ( C ) Tumour-bearing mice from each genotype were injected with pimonidazole prior to sacrifice to measure hypoxic areas within the tumour. 8 µm tumour cryosections were then double stained with anti-pimonidazole antibody (green) to highlight hypoxic areas and anti-PECAM antibody to identify blood vessels. The hypoxic index was quantified relative to PECAM staining using image J software. Bars represent mean relative hypoxic index ± SEM. ( D ) Representative images of pimonidazole detection and PECAM-positive blood vessels in tumour sections. Arrows , blood vessels; Asterisks , pimonidazole-positive staining. Scale bars: A 50 µm; D 200 µm. N = 4 tumours per genotype. NSD: not statistically different, * P

    Article Snippet: 100 µm Z-stacks (stack interval 0.5 µm, 20× magnification) were taken using a Zeiss LSM 510 confocal microscope.

    Techniques: Mouse Assay, Injection, Microscopy, Staining, Software

    CpG-A and CpG-B are distributed in different compartments in PDCs. Purified PDCs were cultured with fluorescent CpG-A (A and C) or CpG-B (E and G). Cells were fixed, stained intracellularly with (A and E) antitransferrin receptor (TfR) or (C and G) anti–LAMP-1 antibodies, and imaged by confocal microscopy. Images were acquired using a ZEISS LSM 510 META confocal microscope. We used CpG-A-Rhodamine green-X and CpG-B-Alexa488. Intensity profiles of the merged channel along three randomly chosen lines ( 1 , 2 , and 3 shown on each merged staining) were analyzed using the profile tools of the Zeiss LSM software. Examples are shown for (B) CpG-A and TfR, (D) CpG-A and LAMP-1, (F) CpG-B and TfR, and (H) CpG-B and LAMP1. The green line represents the intensity of the ISS, whereas the red line represents the intensity of the endosomal marker. Overlap of the two profiles indicates spatial correlation for the occurrence of the two fluorescent signals. Representative data of 5–10 individual donors are shown.

    Journal: The Journal of Experimental Medicine

    Article Title: Properties regulating the nature of the plasmacytoid dendritic cell response to Toll-like receptor 9 activation

    doi: 10.1084/jem.20060401

    Figure Lengend Snippet: CpG-A and CpG-B are distributed in different compartments in PDCs. Purified PDCs were cultured with fluorescent CpG-A (A and C) or CpG-B (E and G). Cells were fixed, stained intracellularly with (A and E) antitransferrin receptor (TfR) or (C and G) anti–LAMP-1 antibodies, and imaged by confocal microscopy. Images were acquired using a ZEISS LSM 510 META confocal microscope. We used CpG-A-Rhodamine green-X and CpG-B-Alexa488. Intensity profiles of the merged channel along three randomly chosen lines ( 1 , 2 , and 3 shown on each merged staining) were analyzed using the profile tools of the Zeiss LSM software. Examples are shown for (B) CpG-A and TfR, (D) CpG-A and LAMP-1, (F) CpG-B and TfR, and (H) CpG-B and LAMP1. The green line represents the intensity of the ISS, whereas the red line represents the intensity of the endosomal marker. Overlap of the two profiles indicates spatial correlation for the occurrence of the two fluorescent signals. Representative data of 5–10 individual donors are shown.

    Article Snippet: Images were acquired using a ZEISS LSM 510 META confocal microscope and a 63×/1.4 N.A. objective, with the pinhole set for a section thickness of 0.8 μm (pinhole set to 1 airy unit in each channels).

    Techniques: Purification, Cell Culture, Staining, Confocal Microscopy, Microscopy, Software, Marker

    The secondary structure of ISS is regulating their intracellular localization in human PDCs. (A and B) Purified PDCs were cultured with fluorescent ISS for 90 min. Cells were fixed, stained intracellularly with antitransferrin receptor (TfR) or anti-LAMP1 antibodies, and imaged by confocal microscopy. We used CpG-A ss-Rhodamine green-X from the same preparation as CpG-A used in Fig. 4 and CpG-B-Alexa488 premixed with PMXB for 30 min. Images were acquired using a ZEISS LSM 510 META confocal microscope. (B) Between 100 and 200 cells were analyzed from three donors for colocalization between the ODN and either transferrin receptor (TfR) or LAMP-1 (LP1).

    Journal: The Journal of Experimental Medicine

    Article Title: Properties regulating the nature of the plasmacytoid dendritic cell response to Toll-like receptor 9 activation

    doi: 10.1084/jem.20060401

    Figure Lengend Snippet: The secondary structure of ISS is regulating their intracellular localization in human PDCs. (A and B) Purified PDCs were cultured with fluorescent ISS for 90 min. Cells were fixed, stained intracellularly with antitransferrin receptor (TfR) or anti-LAMP1 antibodies, and imaged by confocal microscopy. We used CpG-A ss-Rhodamine green-X from the same preparation as CpG-A used in Fig. 4 and CpG-B-Alexa488 premixed with PMXB for 30 min. Images were acquired using a ZEISS LSM 510 META confocal microscope. (B) Between 100 and 200 cells were analyzed from three donors for colocalization between the ODN and either transferrin receptor (TfR) or LAMP-1 (LP1).

    Article Snippet: Images were acquired using a ZEISS LSM 510 META confocal microscope and a 63×/1.4 N.A. objective, with the pinhole set for a section thickness of 0.8 μm (pinhole set to 1 airy unit in each channels).

    Techniques: Purification, Cell Culture, Staining, Confocal Microscopy, Microscopy

    Cytosolic N1IC can interact with CARMA1 in a bifluorescence complementation assay . Jurkat T cells were transfected with various constructs of nuclear and/or cytosolic proteins and their physical association was assessed microscopically by their ability to reconstitute two halves of a yellow fluorescent protein reporter. Twenty-four hours after transfection, cells were plated onto glass bottom culture dishes, stimulated with anti-human CD3ε and anti-human CD28, and fluorescent images of live cells were captured using a Zeiss LSM 510 confocal microscope. (A) Nuclear proteins, cFos and cJun, known to interact in the nucleus were co-expressed as a control; (B) NOTCH1 with an additional nuclear localization signal, N1IC–NLS was co-expressed with nuclear cJun; (C) NOTCH1 with an additional nuclear export signal, N1IC–NES, was co-expressed with nuclear cJun; (D) NOTCH1 with an additional nuclear export signal, N1IC–NES was co-expressed with CARMA1, a cytosolic protein; (E) NOTCH1 with an additional nuclear localization signal, N1IC–NLS was CARMA1; (F) NOTCH1 with an additional nuclear export signal, N1IC–NES was expressed alone. Upper panel of all images represent fluorescent channel only; lower panel of all images represent merged fluorescent and DIC images. Data are representative of at least three separate experiments.

    Journal: Frontiers in Immunology

    Article Title: NOTCH1 Can Initiate NF-?B Activation via Cytosolic Interactions with Components of the T Cell Signalosome

    doi: 10.3389/fimmu.2014.00249

    Figure Lengend Snippet: Cytosolic N1IC can interact with CARMA1 in a bifluorescence complementation assay . Jurkat T cells were transfected with various constructs of nuclear and/or cytosolic proteins and their physical association was assessed microscopically by their ability to reconstitute two halves of a yellow fluorescent protein reporter. Twenty-four hours after transfection, cells were plated onto glass bottom culture dishes, stimulated with anti-human CD3ε and anti-human CD28, and fluorescent images of live cells were captured using a Zeiss LSM 510 confocal microscope. (A) Nuclear proteins, cFos and cJun, known to interact in the nucleus were co-expressed as a control; (B) NOTCH1 with an additional nuclear localization signal, N1IC–NLS was co-expressed with nuclear cJun; (C) NOTCH1 with an additional nuclear export signal, N1IC–NES, was co-expressed with nuclear cJun; (D) NOTCH1 with an additional nuclear export signal, N1IC–NES was co-expressed with CARMA1, a cytosolic protein; (E) NOTCH1 with an additional nuclear localization signal, N1IC–NLS was CARMA1; (F) NOTCH1 with an additional nuclear export signal, N1IC–NES was expressed alone. Upper panel of all images represent fluorescent channel only; lower panel of all images represent merged fluorescent and DIC images. Data are representative of at least three separate experiments.

    Article Snippet: Stained cells were visualized with a Zeiss LSM 510 Meta Confocal Microscope, using a 63× oil immersion objective.

    Techniques: Transfection, Construct, Microscopy