Zymosan A Texas Red particles were rinsed with PBS, resuspended in culture medium, sonicated and mixed for different periods of time (0, 1, 4 and 8 hours) with Raw cells cultured on coverslips or B-lymphocytes that were transferred to a Poly-L lysine (Sigma-Aldrich, St. Louis, MO) coated coverslips. Cells were rinsed with PBS, fixed with 4% formaldehyde and mounted on slides. Cells were viewed with a Zeiss 510 Confocal Laser Scanning Microscope and images were captured using Zen2010 software.
510 confocal laser scanning microscope
Manufactured by Zeiss
Sourced in Germany
The Zeiss 510 confocal laser scanning microscope is a high-performance imaging system designed for advanced microscopy. It utilizes a laser source to provide illumination and a scanning mechanism to capture detailed, high-resolution images of samples. The 510 model is capable of performing confocal fluorescence microscopy, allowing for the visualization and analysis of specific cellular structures and processes.
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Lab products found in correlation
Poly l lysine (pll), by Merck Group (1 mentions)
Collagenase type 1, by Merck Group (1 mentions)
Superblock pbs, by Thermo Fisher Scientific (1 mentions)
Gelatin, by Jackson ImmunoResearch (1 mentions)
Triton x 100, by Bio-Rad (1 mentions)
Bovine serum albumin (bsa), by Jackson ImmunoResearch (1 mentions)
Gelatin, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Fitc conjugated goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Live dead baclight bacterial viability kit, by Thermo Fisher Scientific (1 mentions)
Plan apochromat 40 1.3 na, by Zeiss (1 mentions)
Prolong gold, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 546, by Thermo Fisher Scientific (1 mentions)
Anti ha, by Santa Cruz Biotechnology (1 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
Goat anti rabbit alexa 488, by Thermo Fisher Scientific (1 mentions)
Vectashield without dapi, by Vector Laboratories (1 mentions)
Goat anti mouse alexa 488, by Thermo Fisher Scientific (1 mentions)
Plan apochromat objective, by Zeiss (1 mentions)
27 protocols using 510 confocal laser scanning microscope
1
Zymosan A Texas Red Particle Uptake
2
Visualizing NMJ Microtubules in Muscle Fibers
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The flexor digitorum brevis (FDB) muscles were harvested bilaterally from anesthetized WT and mdx mice. Single myofibers were enzymatically isolated in DMEM supplemented with 0.2 % FBS, 1 µl/ml gentamicin, and 4 mg/ml type I collagenase (Sigma, C0130) for 1 h at 37 °C as previously described [27 (link)].
Myofibers were plated on extracellular matrix (ECM; Sigma E1270)-coated imaging dishes (Matek, P35G-1.0-14-C), fixed with 4 % paraformaldehyde, permeabilized with 0.1 % Triton X-100 in PBS, blocked in Superblock PBS (Thermo Scientific), stained with BTX-594 (Molecular Probes, B13423) and then labeled with an antibody to α-tubulin conjugated to Alexa Fluor 488 (anti-mouse; Invitrogen 32-2588). Digital images were obtained using a Zeiss 510 confocal laser-scanning microscope. Laser intensity was adjusted on a sample-to-sample basis to maximize the amount of microtubules that are visualized. A 14-image Z-stack was taken at 1 µm intervals to account for total depth of the NMJ, and Image-J (NIH) was used to form a composite image. Background was subtracted uniformly, and the image was transformed into a binary image. The motor-endplate was outlined and the image cropped to isolate microtubule labeling at the NMJ. Total area of pixels at the endplate of myofibers was then quantified in Image J.
Myofibers were plated on extracellular matrix (ECM; Sigma E1270)-coated imaging dishes (Matek, P35G-1.0-14-C), fixed with 4 % paraformaldehyde, permeabilized with 0.1 % Triton X-100 in PBS, blocked in Superblock PBS (Thermo Scientific), stained with BTX-594 (Molecular Probes, B13423) and then labeled with an antibody to α-tubulin conjugated to Alexa Fluor 488 (anti-mouse; Invitrogen 32-2588). Digital images were obtained using a Zeiss 510 confocal laser-scanning microscope. Laser intensity was adjusted on a sample-to-sample basis to maximize the amount of microtubules that are visualized. A 14-image Z-stack was taken at 1 µm intervals to account for total depth of the NMJ, and Image-J (NIH) was used to form a composite image. Background was subtracted uniformly, and the image was transformed into a binary image. The motor-endplate was outlined and the image cropped to isolate microtubule labeling at the NMJ. Total area of pixels at the endplate of myofibers was then quantified in Image J.
3
Visualizing Cilia in Mouse and Xenopus
4
Immunofluorescence Analysis of Glycosylation Markers
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1.5 × 105 cells were plated on 12 mm diameter glass coverslips. 48 h later, cells were vehicle-treated or treated with 7.5 mM GlcN for 24 h. Cells were fixed for 20 min with 3% paraformaldehyde (Sigma) in PBS containing 0.9 mM CaCl2 and 0.5 mM MgCl2 (PBS-CM) at room temperature, washed twice in 50 mM NH4Cl in PBS-CM and twice in PBS-CM. Cells were permeabilized for 5 min in 0.5% Triton-X100 (Bio-Rad) in PBS-CM and incubated for 30 min in 0.5% gelatin (Sigma) in PBS-CM. Cells were then incubated for 1 h with the primary antibodies (anti-E-cadherin 1:100, anti- β-catenin 1:100) diluted in 0.5% BSA (Sigma) in PBS. After three washes with 0.2% gelatin in PBS-CM cells were incubated with the second primary antibodies (anti-calnexin 1:100, anti-GS28 1:50) for 1 h. After three washes with 0.2% gelatin in PBS-CM cells were incubated for 20 min with the appropriate rhodamine- or fluorescein-tagged goat anti-mouse or anti-rabbit antibody (Jackson ImmunoResearch, West Grove, PA), diluted 1:50 in 0.5% BSA in PBS. After final washes with PBS, the coverslips were mounted on a microscope slide and examined with a Zeiss 510 confocal laser scanning microscope. Samples were observed by three investigators, without knowledge of the experimental conditions.
5
Immunofluorescent Labeling of Tyrosine Hydroxylase
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Acutely isolated cells on glass coverslips were rinsed twice with phosphate-buffered saline and fixed with 4% paraformaldehyde for 40 min at room temperature. After fixation, these cells were washed with phosphate-buffered saline and then incubated in phosphate-buffered saline containing 2% normal goat serum and 0.1% Triton X-100 for 60 min at room temperature. Cells were then incubated for 2 hours in phosphate-buffered saline containing tyrosine hydroxylase antibodies (diluted 1 : 1000), 2% normal goat serum, and 0.1% Triton X-100. After rinsing three times with phosphate-buffered saline, they were incubated with fluorescence isothiocvanate (FITC)-conjugated goat anti-mouse IgG (Molecular probes) diluted 1 : 1000 in phosphate-buffered saline at room temperature for 1 h. Excess fluorescent antibodies were removed by washing three times with phosphate-buffered saline. Fluorescence images were then obtained using a Zeiss 510 confocal laserscanning microscope (excitation at 488 nm; emission at 505-545 nm). Detailed descriptions of morphologies and staining results have been previously reported [33 (link)].
6
Biofilm Formation and Serum Effects
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To obtain biofilms, as previously described (48 (link)), overnight cultures of NTHi were resuspended in 5 ml equilibrated (37°C, 5% CO2) brain heart infusion broth supplemented with 2 μg NAD/ml and 2 μg heme/ml (sBHI) so that the optical density at 490 nm was 0.65. The bacteria were diluted 1:6 in equilibrated sBHI in a 50-ml nonclosed sterile conical tube and incubated at 37°C in 5% CO2. After 3 h, bacteria were diluted 1:2,500 in equilibrated sBHI, and 200 μl of the bacterial suspension was added to each well of an 8-well chambered glass slide (catalog number 155411; Nunc Lab-Tek) for an overnight incubation at 37°C in 5% CO2, during which biofilms formed. Medium was refreshed for an additional 8 h of incubation, after which immunized or naive serum pools (serum pools from the adult chinchillas used in the in vivo study diluted 1:50 in sBHI; see below) were added to the chambers. After 16 h at 37°C in 5% CO2, biofilms were washed with sterile saline, incubated with a Live/Dead BacLight bacterial viability kit (catalog number L7007; Invitrogen), and fixed for 1 h at room temperature with 10% formalin. Biofilms were immediately viewed on a Zeiss 510 confocal laser scanning microscope, and images were analyzed with COMSTAT software (49 (link)).
7
Fluorescent Labeling of Neural Tissue
8
Visualization of Cilia and Notochord in Embryos
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Embryos were fixed and stained as described by Dent et al.31 . The mouse monoclonal anti-acetylated α-tubulin antibody was used to visualized ciliated cells32 (link). The rabbit anti-Xenopus Cetn antibody (anti-XlCetn) was supplied by Sergie Sokol20 (link). The rabbit anti-human Cetn1 antibody (anti-HsCetn1) was purchased from Sigma. The anti-HsCetn1 antibody is directed against the C-terminal 15 amino acids of Cetn1; a similar sequence exists at the C-termini of X. laevis Cetn2 (13/15 identical) and Cetn4 proteins (12/15 identical), but is absent from the Cetn3 (5/15 identical)(data not shown). Notochord was visualized using the mouse monoclonal anti-keratin sulfate antibody MZ15 obtained from the Developmental Studies Hybridoma Bank33 (link). Immunoperoxidase-stained explants were bleached before staining while immunofluorescently-labeled embryos were not. Fluorescent images were collected using a Zeiss 510 Confocal Laser Scanning Microscope.
9
Immunofluorescence Microscopy of HA-tagged and GRK5 Proteins
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NRVMs or AVMs washed three times in ice cold PBS and fixed in 3% paraformaldehyde (PFA) for 10 min. Then the cells were permeabilized with 0.2% Triton X-100 for 2 min. After three washes in PBS, the cells were incubated with 1% BSA for 30 min and then incubated with an anti-HA (Santa Cruz Biotechnology, 1:200) and anti-GRK5 (Santa Cruz Biotechnology, 1:200), respectively, diluted in 1% BSA. Next, the cells were incubated with the respective secondary antibodies: a mouse monoclonal to reveal the HA-tag (Alexa Fluor 647; Thermo Fisher Scientific, 1:200) and a rabbit polyclonal to reveal GRK5 (Alexa Fluor 546; Thermo Fisher Scientific 1:200). The fluorescent data sets were visualized with a Zeiss 510 confocal laser scanning microscope and analysed by LSM 510 software.
10
Immunocytochemistry and BiFC Analysis of PS1 in HEK 293T Cells
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HEK 293T cells were transfected in 8-well chambered slides (Millipore) coated with polyornithine. At 24 hr post-transfection, cells were washed twice with 1× PBS and fixed for 20 minutes with 4% PFA. Fixed cells were washed with 1× TBS four times, blocked with 1× TBS 3% goat serum/0.25% Triton-X 100 for 30 minutes, then incubated at 4 °C overnight with a polyclonal anti-calnexin antibody (1:1000 dilution; Cell Signaling Technologies, Beverly, MA) and a monoclonal anti-human PS1 specific antibody (1:1000 dilution, provided by Dr. Paul Mathews, NYU), or anti-FLAG (1:1000, Sigma). After overnight incubation, fixed cells were washed in 1× TBS 1% goat serum, incubated with secondary antibody (1:500 dilution; Alexa 488 goat anti-rabbit, Alexa 488 goat anti-mouse and Alexa 594 goat anti-mouse; Invitrogen) for 1 hr at RT, and then washed with 1× Tris buffer before mounting in hard set Vectashield without DAPI (Vector Laboratories). For the BiFC analysis, the experiment was repeated in 4 iterations and at least two regions of interest per iteration were captured. For immunocytochemistry, the experiment was repeated in 3 iterations, and 3 regions of interest per iteration were imaged. Both BiFC and immunocytochemistry experiments were captured on a Zeiss 510 confocal laser-scanning microscope using a 40×/1.4 N.A. Plan-Apochromat objective.
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