Organoids in matrigel were fixed for 20 min with a 4% paraformaldehyde (PFA) solution and made permeable for 10 min with 0.1% Triton X-100 in phosphate-buffered saline (PBS) at room temperature. Then organoids were incubated with a specific mouse monoclonal Ab raised against CD45 and cytokeratin (1:1000 in blocking solution, 3% BSA in TBS-Tween 0.1%, Sigma) for 2 h at 37 °C followed by revelation using Alexa Fluor 633-conjugated anti-rabbit immunoglobulin (Ig)G antibodies and Alexa Fluor 488-conjugated anti-rabbit IgG antibodies, respectively (Jackson Immunoresearch Laboratories, West Grove, PA, USA) at a dilution of 1:1000 for 1 h. The fluorescence was analyzed by an LSM-410 Zeiss confocal microscope.
Lsm 410 confocal microscope
Manufactured by Zeiss
Sourced in United States
The Zeiss LSM 410 is a confocal microscope that allows for high-resolution imaging of biological samples. It uses a focused beam of light to scan the sample, capturing detailed images of individual optical sections within the specimen.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488 conjugated anti rabbit igg antibody, by Jackson ImmunoResearch (2 mentions)
Mountant permafluor medium, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488 conjugated goat anti rabbit igg, by Thermo Fisher Scientific (2 mentions)
Fitc labeled goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Ar n 20, by Santa Cruz Biotechnology (1 mentions)
Texas red phalloidin, by Thermo Fisher Scientific (1 mentions)
Axiovision 4, by Zeiss (1 mentions)
Axiocam mrc5, by Zeiss (1 mentions)
Image pro plus, by Media Cybernetics (1 mentions)
Fluo 4 am dye, by Thermo Fisher Scientific (1 mentions)
Muscarine, by Merck Group (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Alexa fluor 488 and 568, by Thermo Fisher Scientific (1 mentions)
Ab56416, by Abcam (1 mentions)
Nb100 2220, by Abcam (1 mentions)
Bml pw8810, by Enzo Life Sciences (1 mentions)
Cytofix cytoperm solution, by BD (1 mentions)
Primary antibody for pakt, by Cell Signaling Technology (1 mentions)
Axio observer z1 fluorescent microscope, by Zeiss (1 mentions)
Anti map2 antibody, by Santa Cruz Biotechnology (1 mentions)
14 protocols using lsm 410 confocal microscope
1
Immunofluorescent Staining of Organoids
2
Immunofluorescence analysis of SIRT1 in HepG2 cells
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HepG2 cells growing in 24-well plates at the density of 10 × 103 cells/well were treated as described above, fixed using PBS 4% paraformaldehyde for 10 min and then permeabilized for 10 min with PBS 0.1% Triton X-100. Immunostaining was carried out by O/N incubation at 37 °C with specific antibodies against SIRT1 (1:1000; Sigma Aldrich). Cells were then incubated with secondary antibodies conjugated to Alexafluor 488 (1:1000) for 1 h at room temperature. Cells were stained for 2 min at room temperature by 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI, 5 µ/mL), a nuclear and chromosome counterstain that emits blue fluorescence upon binding to AT regions of DNA. The cells were analyzed by an LSM-410 Zeiss confocal microscope (Carl Zeiss Microscopy Ltd., Cambridge, UK).
3
Immunofluorescence Staining of Organoids
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Organoids in matrigel were fixed for 20 min with a 4% paraformaldehyde (PFA) solution and made permeable for 10 min with 0.1% Triton X-100 in phosphate-buffered saline (PBS) at room temperature. Then organoids were incubated with a specific mouse monoclonal Ab raised against Ido-1 (1:1000 in blocking solution, 3% BSA in TBS-Tween 0.1%, Sigma) for 2 h at 37 °C followed by revelation using Alexa Fluor 488-conjugated anti-rabbit IgG antibodies (Jackson Immunoresearch Laboratories, West Grove, PA, USA) at a dilution of 1:1000 for 1 h. The fluorescence was analyzed by an LSM-410 Zeiss confocal microscope.
4
Immunofluorescent Staining of Cells
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The immunofluorescent staining of cells grown on glass slides was performed as described elsewhere [7 (link), 8 (link), 20 (link)]. Cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 and incubated at 4°C overnight with rabbit polyclonal antibodies against TIN2 [20 (link)], γ-H2AX (i.e., phosphorylated-H2AX) (Upstate), or AR (AR-N20; Santa Cruz), or mouse monoclonal antibodies against AR (AR-414; Santa Cruz), AR-V7 (AG10008; Precision) or pATM (10H11-E12, which detects phosphorylation of ATM at serine 1981; Cell Signaling). Cells were then washed and stained with FITC-labeled goat-anti-rabbit-IgG and/or Texas Red-labeled goat-anti-mouse-IgG (Molecular probes) secondary antibodies [7 (link), 8 (link)]. Images of cells were acquired on an LSM-410 confocal microscope (Zeiss). Labeled foci were counted in enlarged photographs.
5
Subcellular Localization of DnaJC18 Protein
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CVI (kidney cell line) and GC2 (germ cell line) cells were maintained in DMEM
medium supplemented with 10% fetal bovine serum and used for the transfection
experiments and. Cells were seeded in Lab-Tek II chamber slide (Nalge Nunc Inc.,
NY, USA) with 2 ×104 cells per chamber in 150 µL of medium. Cells
were transiently transfected with 0.2 µg of each pEGFP-C1 vector (control
plasmid) and pEGFP-DJC-5 (GFP-DnaJC18 fusion construct) using Lipofectamine Plus
reagent (Qiagen, CA, USA) according to the manufacturer’s instruction. The
transfected cells were subjected to localization study with confocal microscopy
after 24 hours of transfection. A Zeiss LSM 410 confocal microscope equipped
with an external krypton/argon laser was used for localization studies. Image
processing was performed using IP Labs (Scanalytics) and Adobe Photoshop 7.0
(Park & Cheon, 2015 (link)).
medium supplemented with 10% fetal bovine serum and used for the transfection
experiments and. Cells were seeded in Lab-Tek II chamber slide (Nalge Nunc Inc.,
NY, USA) with 2 ×104 cells per chamber in 150 µL of medium. Cells
were transiently transfected with 0.2 µg of each pEGFP-C1 vector (control
plasmid) and pEGFP-DJC-5 (GFP-DnaJC18 fusion construct) using Lipofectamine Plus
reagent (Qiagen, CA, USA) according to the manufacturer’s instruction. The
transfected cells were subjected to localization study with confocal microscopy
after 24 hours of transfection. A Zeiss LSM 410 confocal microscope equipped
with an external krypton/argon laser was used for localization studies. Image
processing was performed using IP Labs (Scanalytics) and Adobe Photoshop 7.0
(Park & Cheon, 2015 (link)).
6
Visualizing Motor Endplates and Innervation
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To study the NMJ, motor endplates in diaphragms were visualized using fluorescent-conjugated α-bungarotoxin (BTX) that binds irreversibly to postsynaptic acetylcholine receptors on the skeletal muscle plasma membrane (67 (link)). Motor endplates were visualized with Alexa 594-conjugated α-BTX (Invitrogen) as described (29 (link)). MN axons were visualized in two ways. We generated double tg mice by crossing hSOD1mus tg mice with B6.Cg-tg Hlxb9-gfp1Tmj/j mice expressing eGFP driven by the mouse Hb9 promoter (68 (link)). Dual labeling was also done to visualize MN distal axons and their synaptic terminals in skeletal muscle by immunofluorescent detection of either neurofilament protein or synaptophysin (Table 1 ). The immunofluorescent labeling for neurofilament was used to determine whether the endplates were innervated. Sections were analyzed using a Zeiss Axiphot epifluorescence microscope or an LSM 410 confocal microscope. The BTX staining patterns were used to assess quantitatively endplate structure in age-matched hSODmus tg and non-tg mice. Endplates were scored as innervated or denervated.
7
Examining Protein Stability and Actin Dynamics
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For CHX chase assays, cells expressing shCONT or shHSP90 were treated with 100 μg/ml of CHX for the indicated hours. Western blotting was then performed to determine the half-life of the NAP1 protein. For phalloidin staining, cells were fixed with 3.7% formaldehyde in PBS for 15 min and stained with Texas-red phalloidin (Molecular Probes, Eugene, OR) for 30 min, and then visualized using a Zeiss LSM 410 confocal microscope. In all quantifications, only those cells presenting with free borders were considered, and at least 100 cells from randomly selected fields were evaluated.
8
Evaluating Cardiac Collagen and Sarcomere Integrity
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To evaluate collagen histology, hearts were fixed in 10% formalin for histologic analysis using picrosirius red as previously described.19 Images were captured with brightfield microscopy using the Zeiss Imager D1, connected to a Zeiss AxioCam MRc5 color camera and using AxioVision 40 version 4.7.1.0 acquisition software (Zeiss, Germany) with 10 images per section, from 2 sections per heart. Image analysis was performed using Image Pro Plus (V4.5.1, Media Cybernetics, Bethesda, MD) in a “blinded” manner. For ultrastructural analysis to examine cardiomyocyte sarcomeric integrity, high‐resolution confocal microscopy was performed on fixed tissues labeled with the fluorescent marker wheat germ agglutinin to delineate cardiomyocyte T‐tubule geometry using a Zeiss LSM410 confocal microscope and ×63 NA 1.25 oil‐immersion objective. The t‐tubule images were converted to frequency space using fast Fourier transform, and the peak in the power spectrum corresponding to the sarcomere spacing was estimated by Gaussian fit (“T power”).
9
Calcium-dependent Muscarinic Signaling in Cells
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Wild-type and CaMKK2 KO (clone A5) cells were cultured for 48 h in serum-free media. The culture media was then replaced with a salt-glucose solution containing 114 mM NaCl, 0.22% NaHCO3, 5.29 mM KCl, with or without CaCl2.2H2O (with calcium: 2 mM CaCl2+1 mM BaCl2; without calcium: 3 mM BaCl2), 10 mM HEPES, 10 mM Glucose, 1 mM MgCl2 and supplemented with 5 μM Fluo-4AM dye (ThermoFisher, F14210). The cells were loaded with cell-permeant Fluo-4 for 15 min, washed and then time series images were captured at 30 s interval following 10 μM Muscarine (Sigma, M6532) treatment. Zeiss LSM410 confocal microscope with a controlled humidified atmosphere containing 5% CO2 at 37°C was used to capture time lapse images. The mean Fluo-4 intensity was calculated from time series images using ImageJ time-series analyzer plugin.
10
Immunostaining of Neuronal Markers
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