LGs from male NOD and BALB/c mice at the indicated ages were retrieved following euthanasia; fixed in 4% paraformaldehyde for 3 h, transferred to 30% sucrose and incubated overnight at 4oC. Fixed glands were inserted into OCT compound and flash-frozen on dry ice. The blocks were cryosectioned at 5 μm thickness and mounted onto a glass slide. The sections were then permeabilized with 0.1% Triton X-100 for 10 min and 1% SDS for 5 min. Following this, the sections were blocked with 1% BSA for 1 h and later incubated with primary and secondary antibodies for a period of 1 h each at 37oC. Between each incubation, the slides were washed with PBS three times for 5 min each. Finally, the samples were mounted with ProLong anti-fade and imaged using a Zeiss LSM 800 confocal fluorescence microscope (Zeiss, Thornwood, NY). Average Mean gray values of 3 random acini from 3 randomly selected images obtained from each of 3 mice in each age group was measured using ImageJ software (National Institutes of Health, http://imagej.nih.gov/ij ) to quantify the expression level of different proteins tested.
Lsm 800 confocal fluorescence microscope
Manufactured by Zeiss
Sourced in Germany
The LSM 800 is a confocal fluorescence microscope manufactured by Zeiss. It is designed to capture high-resolution, three-dimensional images of fluorescently labeled samples. The instrument utilizes a laser-scanning technology to excite fluorophores and detect the emitted light, enabling optical sectioning and the creation of detailed, depth-resolved images.
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Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (4 mentions)
Chambered coverglass, by Thermo Fisher Scientific (1 mentions)
Normal goat serum (ngs), by BioGenex (1 mentions)
Lsm image examiner software, by Zeiss (1 mentions)
Prolong glass antifade mountant, by Thermo Fisher Scientific (1 mentions)
Lc3 antibody, by Cell Signaling Technology (1 mentions)
Ubiquitin antibody, by Proteintech (1 mentions)
Mitotracker orange cm h2tmros, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 labeled phalloidin, by Thermo Fisher Scientific (1 mentions)
10 protocols using lsm 800 confocal fluorescence microscope
1
Quantification of Protein Expression in Mouse Lacrimal Glands
2
Quantifying Oxidative Stress in PA-treated AML12 Cells
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PA-treated AML12 cells with or without UMB were used for the evaluation of the effect of UMB on oxidative stress. After 24 h of treatment, 10 µM 2',7'-Dichlorofluorescin diacetate (DCF-DA) staining solution (Thermo Fisher Scientific) was added to each well at 37℃ for 30 min. The cells were then fixed with 3.7% formaldehyde at RT for 30 min, followed by staining and mounting with a DAPI-containing mounting solution. Stained images were captured using the ZeissLSM800 confocal fluorescence microscope (Carl Zeiss).
3
Immunofluorescence Assay of Huh7 Cells
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Cell immunofluorescence assay was performed as previously described (Khakpooret al., 2009; Gao et al., 2015 (link)). Briefly, treated Huh7 cells were cultured on a chambered coverglass (Thermo Fisher Scientific) and then fixed with 4% paraformaldehyde for 15 min at RT. The samples were permeabilized with 0.1% Triton X-100 for 5 min at RT and then blocked with 3% BSA (in PBS) for 2 h. Afterward, they were incubated with the indicated primary antibodies (2 h at RT) and fluorophore-conjugated secondary antibodies (1 h at RT) in 3% BSA and then stained with DAPI. The samples were observed using a Zeiss LSM 800 confocal fluorescence microscope. Images were adjusted for brightness and contrast and collocated into figures by using Photoshop CS8.
4
Retinal Vasculature Imaging in Mice
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Mice were killed and their eyes were enucleated in phosphate-buffered saline (PBS). The eyes were then fixed in 4% paraformaldehyde for 1 h at 4 °C, after which the retinas were removed, washed with PBS, and permeabilized with PBS containing 1% Triton X-100 for 1 h. The retinas were subsequently incubated overnight in 0.1% Tween and 10% normal goat serum (BioGenex, Fremont, CA) in PBS at 4 °C, followed by incubation for 1 h in Alexa Fluor 597-conjugated isolectin GS-IB4 solution (1:100) at 4 °C. Flat-mounted retinas were analyzed using an LSM800 confocal fluorescence microscope (Carl Zeiss Microscopy, Cambridge, MA). Quantification was performed by researchers unaware of the mouse’s genotype.
5
Peptide Uptake in A549 and HeLa Cells
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A549 and HeLa cells were cultured in DMEM high glucose media supplemented with 10% fetal bovine serum, 1% Penstrep, 0.2% Amphotericin B. The cells were grown overnight at 37 °C incubator with 5% CO2. A549 and HeLa cells were seeded at a density of 3×105 cells in 35 mm glass-bottomed dish and kept overnight prior to cell imaging studies. After 24 h, the cells were washed twice with warm DMEM media, incubated at 37 °C with 5 µM peptides for 60 min, and then pretreated with 5 μM probe for 15 min. Images were taken using the Zeiss LSM 800 confocal fluorescence microscope.
6
Immunohistochemical analysis of frozen tissue
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Tissues immersed in OCT were quickly frozen in liquid nitrogen and cut into 7-µm-thick sections. Frozen tissue sections were fixed in cold acetone for 10 min at −20°C, blocked with 5% BSA and 1:100 Fc-blocker in PBS, and stained with biotin-IgD, FITC-labeled anti-GL7, and PE-labeled anti-CD4, followed by Alex 650 dye-labeled avidin. After each step, the slides were washed at least three times with PBS. Coverslips were mounted on slides using an antifade kit (BOSTER) and then examined using a Zeiss LSM 800 confocal fluorescence microscope. The images were processed with LSM Image Examiner software (Zeiss).
7
Immunofluorescent Staining of Mitochondrial ROS
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Immunofluorescent staining was performed based on the previous study (Han et al., 2019). In brief, 1–2 × 104 cells were seeded on Nunc™ Lab‐Tek™ II four‐well chamber slide™ w/ removable wells (Thermo Scientific). Cells were washed with PBS and stained with 250 nM MitoSpy™ Orange CMTM ROS dye (BioLegend, San Diego, CA) for 20 min at 37°C according to the manufacturing protocols. Then cells were fixed with 4% paraformaldehyde (PFA) and permeabilized with 0.1% Triton X‐100. After permeabilization, cells were stained with 1 μg/mL DAPI (Thermo Scientific), then mounted with ProLong™ Glass Antifade Mountant (Thermo Scientific). Prepared samples were visualized with a 63× oil immersion objective lens of an LSM800 confocal fluorescence microscope (Carl Zeiss, Oberkochen, Germany).
8
Immunostaining of Macrophages
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BMDMs (4 × 105 cells/well) were seeded into 12-well plates and incubated with or without oxLDL or AMY. After a 24 h-incubation, cells were washed twice with phosphate buffer saline, then fixed with formaldehyde and permeabilized by 0.2% Triton X-100. The antibody was added to each slide, shaken slightly on a shaker overnight at 4°C. Then, the slides were incubated with anti-rabbit IgG ab and DAPI for 2 h and 2 min, respectively. Finally, cells were observed using a LSM800 confocal fluorescence microscope (ZEISS, Germany). Frozen slices were used to detect lymphatic vessels of heart atrium in the same way.
9
Immunofluorescence Assay for LC3 and Ubiquitin
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Cells were fixed in 4% paraformaldehyde in PBS for 10 min and permeabilized with 0.1% Triton X-100 in PBS for 30 min at room temperature. After blocking in 10% normal goat serum, cells were incubated with rabbit polyclonal microtubule-associated protein 1 light chain 3 (LC3) antibody (1:200; Cell Signaling Technology [CST], Danvers, MA, USA) and ubiquitin antibody (1:100; Proteintech, Rosemont, IL, USA) overnight at 4 °C followed by incubation with Cy3-labeled and Alexa Fluor 488-labeled secondary antibody (1:1000; Beyotime, Shanghai, China) for 1 h at room temperature. Nuclei were stained in the dark with DAPI. Fluorescence images were visualized using the LSM 800 confocal fluorescence microscope (Zeiss, Oberkochen, Germany).
10
Meiotic Spindle and Chromosome Imaging
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To assess meiotic spindle and chromosome configurations after drug treatment, individual oocytes were fixed for 60 minutes at 37 °C in 100 mM HEPES (pH 7) titrated with KOH, 50 mM EGTA (pH 7) titrated with KOH, 10 mM MgSO 4 , 2% formaldehyde (methanol-free), and 0.2% Triton X-100 on the basis of a previously published protocol ). After fixation, oocytes were rinsed in phosphate-buffered saline supplemented with 0.1% Triton X-100 (PBT) and kept in PBT overnight at 4 °C. For microtubule staining, oocytes were exposed to an anti-α-tubulin antibody (rat monoclonal MCA78G, Bio-Rad, Hercules, CA) overnight at 4 °C and an Alexa-Fluor-633-labeled goat anti-rat antibody (A-21094, Thermo Fisher Scientific, Waltham, MA) for 2 hours in the dark and at room temperature. Chromatin was stained using DAPI (D1306, Thermo Fisher Scientific) and actin filaments using Alexa-Fluor-488 labeled phalloidin (A12379, Thermo Fisher Scientific) for 1 hour in the dark at room temperature. Incubations with primary and secondary antibodies and fluorescent dyes were performed in PBT supplemented with 3% bovine serum albumin. For mitochondria visualization, live cells were treated with the fluorescent probe MitoTracker Orange CM-H2TMRos (M7511, Thermo Fisher Scientific) for 1 hour in the dark at 37 °C before fixation. Samples were imaged using a Zeiss LSM 800 confocal fluorescence microscope.
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