Cryosections of heart valve tissue were fixed with 4% paraformaldehyde and incubated overnight at 4°C with anti-Staphylococcus aureus antibody (1:100; anti-Staphylococcus aureus antibody 704, ab37644, abcam, Germany). Indirect immunofluorescence was performed using secondary antibodies conjugated with Alexa Fluor 488 (1:500; Invitrogen, Life Technologies GmbH). Cell nuclei were stained with hexidium iodide (1:500; Molecular Probes, Life Technologies GmbH, Darmstadt, Germany). The tissues were analyzed with a Zeiss LSM-5 Pascal confocal laser scanning microscope (Carl Zeiss, Jena, Germany).
Lsm 5 pascal laser scanning confocal microscope
Manufactured by Zeiss
Sourced in Germany
The LSM 5 PASCAL is a laser scanning confocal microscope manufactured by Zeiss. It is designed to provide high-resolution imaging of samples by using a focused laser beam to scan the specimen and collect the resulting fluorescence or reflected light signals. The LSM 5 PASCAL is capable of producing detailed, three-dimensional images of biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Ab150077, by Abcam (2 mentions)
μ slide vi0, by Ibidi (2 mentions)
Eclipse ti s, by Nikon (2 mentions)
Hexidium iodide, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Streptavidin alexa fluor 594 conjugate, by Thermo Fisher Scientific (1 mentions)
Vectashield mounting medium, by Vector Laboratories (1 mentions)
Occludin, by Thermo Fisher Scientific (1 mentions)
Anti claudin 5, by Thermo Fisher Scientific (1 mentions)
Dnase 1, by Sangon (1 mentions)
Dmem f12 medium, by Wisent (1 mentions)
Wga fitc, by Thermo Fisher Scientific (1 mentions)
Soybean trypsin inhibitor, by Sangon (1 mentions)
Collagenase p, by Roche (1 mentions)
Lr clonase 2, by Thermo Fisher Scientific (1 mentions)
Pascal imaging software, by Zeiss (1 mentions)
Hoechst 33258, by Zeiss (1 mentions)
Lsm 510 confocal laser scanning microscope, by Zeiss (1 mentions)
Sudan black b, by Fujifilm (1 mentions)
Hoechst 33258, by Polysciences (1 mentions)
17 protocols using lsm 5 pascal laser scanning confocal microscope
1
Immunofluorescence Analysis of Staphylococcus aureus in Heart Valve Tissue
2
Immunofluorescent Labeling of LR11 and CD9
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Cells were fixed in 100% methanol at −20 °C for 15 min. Fixed cells were incubated with a biotinylated mAb against LR11 (R14) followed by an Alexa Fluor 594 streptavidin conjugate (Invitrogen). Next, the cells were incubated with a fluorescein isothiocyanate-conjugated anti-CD9 antibody M-L 13. Cells were mounted with VECTASHIELD Mounting Medium (Vector Laboratories, Burlingame, CA, USA). Slides were examined with the Zeiss LSM5 PASCAL confocal laser scanning microscope (Carl Zeiss, Oberkochen, Germany).
3
Immunohistochemical Characterization of MBECs
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For immunohistochemical characterization, MBECs were stained with anti-claudin-5, occludin, ZO-1 (Invitrogen Corporation, Waltham, MA, USA), or von Willebrand factor antibodies. Astrocytes were stained with anti-GFAP antibody (Progen Scientific Ltd., Mexborough, UK). All primary antibodies were used at a dilution of 1:100. As secondary antibodies Alexa Fluor 488 conjugated donkey anti-rabbit and anti-mouse immunoglobulins (both from Invitrogen Corporation) were used at a dilution of 1:1000. The source and catalogue number of antibodies is listed in Table S1 . To counterstain cell nuclei TO-PRO-3 Iodide (Invitrogen Corporation) was used at a dilution of 1:400. Cultured cells were fixed in 3% paraformaldehyde in PBS for 10 min and permeabilized with 0.1% Triton X-100 for 10 min. Cells were blocked with 3% bovine serum albumin and were incubated with primary antibodies overnight at 4 °C. After washing, cells were incubated for 1 h at room temperature with secondary antibodies and TO-PRO-3. Cells were washed three times with PBS, and preparations were mounted with Gel Mount (Biomeda, Foster City, CA, USA) and staining was examined using a Zeiss LSM 5 Pascal Confocal laser scanning microscope (Carl Zeiss AG, Oberkochen, Germany).
4
Confocal Microscopy Imaging Protocol
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A Zeiss LSM5 Pascal Confocal Laser Scanning Microscope (Carl Zeiss Jena, Germany) equipped with HeNe and Argon laser were used in this study. For experiments in which subcellular distribution was the main aim, the microscope and laser setting for each acquisition were adjusted to achieve maximal image quality for any individual cell. For experiments in which fluorescence intensity was used as a measure of protein expression, the imaging protocol was fixed in order to allow quantitative comparisons of relative fluorescence intensity among comparable samples.
5
Isolation and Characterization of Pancreatic Acinar Cells
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The pancreatic acinar cells were isolated from 22–30 g male BALB/c mice using collagenase P (Roche) digestion, DNase I (Sangon Biotech), and soybean trypsin inhibitor (Sangon Biotech) [22 (link)]. Cells were resuspended in DMEM/F12 medium (Wisent) supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, and 10% FBS. After an overnight incubation, “contaminated” cells adhered were discarded, and suspended acinar cells were collected and treated with 10 mM alloxan (Sangon Biotech) for 10 min. For lineage tracing, cells were labeled with 5 μg/mL WGA-FITC (Thermo Fisher Scientific, Waltham, MA, USA) for 24 h and then treated with 100 nM rReg3α. At d 3 and 5, cells were fixed in 4% paraformaldehyde and permeabilized in 0.2% Triton X-100 followed by an incubation with anti-insulin or anti-Ngn3 and Cy5-conjugated secondary antibodies (Table 1 ). Microscopic images were captured using a Zeiss LSM 5 Pascal laser scanning confocal microscope with 630× magnification.
6
Subcellular Localization of NPF2.5
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To determine the subcellular localisation of NPF2.5, the coding sequence of NPF2.5 was transferred to the destination vector pMDC44 (Curtis and Grossniklaus, 2003 (link)) using LR Clonase II (Invitrogen, CA). The vector pMDC44 containing 5′ GFP6 was used for generating a GFP::NPF2.5 construct that was transformed into Col-0 Arabidopsis plants using the floral dip method. Two independent transformations were performed to generate two independent lines. T2 transgenic plants were geminated on ½ MS media under hygromycin selection (25 μg/ml) and grown for 2 weeks. GFP fluorescence was visualized using an LSM5 PASCAL laser-scanning confocal microscope (Carl Zeiss, Jena, Germany) running PASCAL imaging software (version 3.2 SP2, Carl Zeiss) with an excitation of 488 nm and an emission of 505–530 nm. Plasmolysis was conducted on a slide using 10% (w/v) sucrose solution.
7
Brain Slice Glioblastoma Invasion Assay
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The brain slice invasion assay was performed as described previously (Valster et al, 2005 (link)). Animals were maintained and brain slices obtained under a research protocol approved by the Institutional Animal Care and Use Committee of St Joseph's Hospital and Medical Center, Barrow Neurological Institute (Phoenix, AZ, USA). Briefly, 24 h after transfection, 1 × 105 GFP-labelled SNB19 or U87 cells were deposited in 0.5 μl DMEM onto the bilateral putamen of 400 μm thick slices of freshly isolated 4- to 6-week-old mouse brain. Serial Z-sections were collected using a LSM 5 Pascal laser-scanning confocal microscope (Zeiss, Thornwood, NY, USA) and the extent of invasion determined as the maximum depth of invasion of the glioblastoma cells.
8
Immunostaining for Neurodegenerative Markers
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Immunofluorescent staining for MAP2, GFAP, MBP, CD68, p62, ubiquitin, and TDP-43 was carried out on free-floating sections. To improve specific fluorescent signals and reduce autofluorescence, antigen retrieval and Sudan black B treatment was performed as described previously [22 (link)]. Briefly, sections were incubated in 10 mM citrate buffer (pH 6.0) at 90°C for 30 min, followed by incubation in 0.1% Sudan black B (Wako Pure Chemicals) in 70% ethanol for 20 min at RT. The sections were blocked in blocking solution (Block Ace, Snow Brand Milk Products) at RT for 2 h and exposed to primary antibody at 4°C for 60 h. Sections were incubated with Alexa Fluor 488- or 594-conjugated secondary antibody (1:500) at RT for 2 h. Nuclei were stained with Hoechst33258 (0.1 μg/ml; Polysciences Inc., Warrington, PA) at RT for 30 min. All sections were mounted on glass slides and coverslipped with fluoromount (Diagnostic BioSystems, Pleasanton, CA). To analyze the localization of p62, the sections were examined under an LSM 510 laser scanning confocal microscope (Carl Zeiss, Oberkochen, Germany). For triple staining for p62, ubiquitin, and Hoechst33258; p62, TDP-43, and Hoechst33258, the sections were examined under an LSM 5 PASCAL laser scanning confocal microscope (Carl Zeiss).
9
Immunohistochemical Analysis of Caveolin-3
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6-μm-thick cut sections were taken from the deparaffinized process described above and rehydrated positively charged slides for immunohistochemical assay. For antigen retrieval, the sections were kept in Antigen Unmasking Solution (Vector Laboratories) in 90 °C for 20 min and incubated an with anti-cav-3 antibody (1:500 dilution) (ab150077, Abcam, Cambridge, UK) for one night. It was incubated all sections with secondary antibody Alexa 488 (1:1000 dilution) (ab2912, Abcam, Cambridge, UK) and Cy3-conjugated donkey anti-mouse IgG antibody (1:1000 dilution) (ab150077, Abcam, Cambridge, UK) during one h. Finally, the sections were examined using a Zeiss LSM 5 PASCAL laser scanning confocal microscope.
10
Flow Cell Inoculation and Imaging
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Inoculation of flow cells was done by diluting overnight-grown cultures to an OD600 of 0.04 and injecting into a μ-Slide VI0.4 (Ibidi, Martinsried, Germany). To inoculate the flow cell surface, bacteria were allowed to adhere at room temperature for 1 h. Flow of 2% v/v LB (0.02% tryptone, 0.01% yeast extract, 1% NaCl; pH 7.5) containing 20μg/mL ampicillin and 100μM IPTG was initiated at a rate of 7.5 ml/h and continued for 24 h. Confocal images were obtained on a Zeiss LSM 5 PASCAL Laser Scanning Confocal microscope. Images were obtained with a 40X dry objective and were processed using Imaris (Bitplane, Zurich, Switzerland). Quantitative analyses were performed using the COMSTAT software package [108 (link)]. Statistical significance was determined using Oneway ANOVA with Dunnett’s Multiple Comparison test. Two biological replicates were performed in triplicate. Images presented are from one representative experiment.
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