Chondrocytes (5 × 104 cells/well) were inoculated in 24-well plates. After treatment with 10 ng/ml IL-1β for 2 h followed by treatment with L-G ln (0, 5, 10, or 20 mM) for another 24 h, the cells were fixed in 4% paraformaldehyde for 40 min. The cells were permeabilized with 0.4% Triton X-100 (Beyotime) for 25 min and blocked with 20% donkey serum (Shyuanye, China) for 45 min. The cells were incubated with antibodies (1:200) at 4°C for 8 h, followed by incubation for 30 min in the dark with the fluorescent secondary antibody (550037, Alexa Fluor 488, 1:500, ZBNBIO) at 25–30°C. Finally, the nuclei were stained with DAPI (Beyotime) for 6 min. Anti-fluorescence quenching agent was added dropwise. The images of p65, SMAD2/3, aggrecan, collagen II, and MMP-13 staining were captured with a laser confocal microscope (LSM8800, Carl Zeiss, Shanghai, China) and analysed by Image-Pro Plus 6.0 software.
Lsm8800
Manufactured by Zeiss
Sourced in Germany
The LSM8800 is a laser scanning microscope system manufactured by Zeiss. It is designed to provide high-resolution imaging capabilities for various applications in scientific research and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Airyscan confocal microscope, by Zeiss (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Zen blue software, by Zeiss (2 mentions)
Triton x 100, by Beyotime (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
Fluo 4 direct, by Thermo Fisher Scientific (1 mentions)
35 mm glass bottom dishes, by MatTek (1 mentions)
Anti collagen 2, by Abcam (1 mentions)
Alcian blue, by Abcam (1 mentions)
Alexa flour secondary antibody, by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Biosesang (1 mentions)
Panoramic midi, by 3DHISTECH (1 mentions)
Masson s trichrome, by Abcam (1 mentions)
Anti aggrecan, by Abcam (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)
Safranin o, by ScienCell (1 mentions)
Fluorescence microscope, by Zeiss (1 mentions)
8 protocols using lsm8800
1
Chondrocyte Inflammation and L-Glutamine
2
Intracellular Calcium Dynamics Monitoring
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Intracellular calcium levels were monitored using the fluorescent calcium indicator Fluo4-Direct (Invitrogen) according to manufacturer’s instructions. Tet-inducible stable cell lines (wild-type; WT NCX1 and unpalmitoylatable; C739A) were cultured on poly-l-lysine (PLL, Sigma) coated 35mm glass bottom dishes (MatTek Life Sciences). 16-24h after inducing NCX1 expression with tetracycline (1μg/ml), cells were loaded with Fluo4 for 1h at 37°C. Calcium indicator loaded cells were imaged using a Zeiss LSM8800 with Airyscan confocal microscopy (λexcitation = 494 nm, λemission = 520 nm).
3
Histological Analysis of Cartilage Formation
4
Porcine Oocyte Immunofluorescence Staining
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porcine oocytes removed from zona pellucida were fixed in 4% paraformaldehyde for 30 minutes, and infiltrated with 1% Triton X-100/PBS (v/v) for 20 minutes. The oocytes were blocked with PBS containing 1% bovine serum albumin (BSA) at 37°C for 1 h-1.5 h. The oocytes were treated with anti-a-tubulin-FITC antibody (Sigma, F2168, 1:200) and anti-phalloidin-TRITC antibody (Beyotime, C1033, 1:200) for overnight. The oocytes were incubated with the secondary antibody at 37°C for 1.5 h, and was counter-stained with propidium iodide (10 μg/ml) at room temperature for 10 minutes. The primary and secondary antibodies were diluted with 0.1% BSA in PBS. Fluorescence was detected Chromosomal arrangement is performed by a fluorescence microscope (Nikon, Tokyo, Japan) or a confocal microscope (LSM 8800, Zeiss, Oberkochen, Germany). The fluorescence intensity of each oocyte was measured under the same scan settings. Image J software was used to analyse the fluorescence intensity of oocytes, and the mean value was calculated after separating the channels.
5
Immunofluorescence Analysis of Porcine Oocytes
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After removing the zona pellucida by acidic Tyrode solution (pH 2.5), the porcine oocytes were fixed in darkness for 30 min with 4% paraformaldehyde, and permeabilized by 1% Triton X-100/PBS (Phosphate-buffered saline) (v/v) for 20 min. Following permeabilization, the oocytes were blocked with PBS containing 1% bovine serum albumin (BSA) at 37° C for 1 h, porcine oocytes were incubated with primary antibodies (Table 1 ) at 4° C overnight. Then, the oocytes were incubated with secondary antibodies at 37° C for 1.5 h, oocyte nucleus were stained with Hoechst 33342 (10 μg/ml) for 15 min. The primary antibodies and secondary antibodies were diluted by PBS with 0.1% BSA. Oocytes were washed with PBS three times between each step above. Prolong Gold Antifade Mountant (Invitrogen, P36930) was dropped in the center of the slide and the oocytes after the above treatment were transferred to the drop followed by the coverslip on it. Fluorescence was detected by a fluorescence microscope (Nikon, Tokyo, Japan) or confocal microscope (LSM 8800, Zeiss, Oberkochen, Germany).
6
Immunofluorescence Staining of Oocytes and Embryos
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Immuno uorescence staining was performed according with our description previously [20] . Brie y, the oocytes or embryos were removed of zona pellucida with 0.5% acidic Tyrode solution. After washed with PBS/PVA (1 mg/1 mL) for three times, oocytes or embryos were xed with 4% paraformaldehyde (PFA) for 40 min at room temperature. The samples were permeabilized with 1% Triton-X 100/PBS for 20 min and blocked with 2% BSA/PBS for 1 h at RT. Then the samples were incubated overnight at 4 °C with primary antibodies according to manufacturer's recommended dilutions. After being washed with PBS/PVA for three times, samples were stained with secondary antibodies in the dark for 2 h at 37 °C. Then the DNA was stained with 10 µg/mL DAPI for 15 min. The oocytes or embryos were mounted on coverslips in ProLong Diamond Antifade Mountant reagent (Life Technologies, USA). A uorescence microscope (Nikon, Tokyo, Japan) was used to capture uorescent images by setting the same parameters for all groups. The uorescent images for α-tubulin were taken by a confocal laser-scanning microscope (LSM8800, Zeiss, Oberkochen, Germany). The relative uorescence intensity was analyzed by Image J software (National Institutes of Health, Bethesda, MD) according to the previous description.
7
Visualizing GFP-tagged REST Protein in HEK-293 Cells
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HEK-293 human embryonic kidney cells [51 (link)] were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% (v/v) fetal bovine serum (Thermo Fisher Scientific, Waltham, MA, USA) and 1% (v/v) penicillin/streptomycin (Sigma-Aldrich, St. Louis, MO, USA). Cells were cultured in a humidified incubator at 37 °C with 5% CO2. Mycoplasma contamination was screened using bisBenzimide H 33342 trihydrochloride (14533, Sigma-Aldrich, St. Louis, MO, USA) DNA staining. The HEK-293 cells were plated in 35 mm dishes (density 4 × 104 cells per mL) 16 h before transfection. Cells were transiently transfected using X-tremeGENE™ HP DNA Transfection Reagent according to the manufacturer’s instructions (Roche, Basel, Switzerland), with 250 ng of plasmid (Empty, GFP-only, GFP-tagged WT REST or GFP-tagged MT REST). Live viewing was performed 24 h after transfection, using a Zeiss LSM8800 with Airyscan confocal microscope (Zeiss, Oberkochen, Germany). Cells were spiked with 1 in 100,000 Hoechst for co-visualization of nuclear material. The detector of the confocal was the photomultiplier tube (PMT) and allowed detection of the green fluorescence signal through the Argon laser at 488 nm. Images were visualized and processed using the ZEN Blue Software (latest version) provided by Zeiss (Zeiss, Oberkochen, Germany).
8
Live Cell Imaging of Transfected Cells
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Live viewing was performed 48 hours following transfection, using a Zeiss LSM8800 with Airyscan confocal microscope (Zeiss). The detector of the confocal was a photo multiplier tube (PMT) and allowed detection of the green fluorescence signal through the Argon laser at 488 nm. Images were visualized and processed using the ZEN Black Software (latest version) provided by Zeiss (Zeiss).
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