In order to assess the ROS levels, CM‐H2DCFDA (C6827; Invitrogen) was used. Oocytes were incubated in M16 medium containing with 5 μM CM‐H2DCFDA for 30 min at 37°C in 5% CO2 incubator. Following washing three times, oocytes were mounted on a live cell‐imaging dish and covered with mineral oil. Immediately, taking fluorescent images using a Zeiss Laser Scanning Confocal Microscope (LSM 710; Zeiss).
Laser scanning confocal microscope lsm710
Manufactured by Zeiss
Sourced in Germany
The ZEISS laser scanning confocal microscope (LSM710) is a high-performance imaging system designed for advanced microscopy applications. It utilizes laser technology and confocal principles to provide high-resolution, optical sectioning capabilities.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Merck Group (4 mentions)
Prolong diamond antifade, by Thermo Fisher Scientific (3 mentions)
Paraformaldehyde phosphate buffer solution, by Nacalai Tesque (2 mentions)
Cm h2dcfda, by Thermo Fisher Scientific (1 mentions)
Computer controlled filter wheel, by Sutter Instruments (1 mentions)
Iq software, by Oxford Instruments (1 mentions)
Neo scmos camera, by Oxford Instruments (1 mentions)
Ab108986, by Abcam (1 mentions)
Ab150075, by Abcam (1 mentions)
Horse serum, by Thermo Fisher Scientific (1 mentions)
Cm3050, by Leica (1 mentions)
Dapi fluoromount g, by Southern Biotech (1 mentions)
Cy3 conjugated goat anti rat igg secondary antibody, by Beyotime (1 mentions)
Zen 2012 imaging software, by Zeiss (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594 conjugated anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 conjugated anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Plan apochromat 20 0, by Zeiss (1 mentions)
Alexa fluor 488 and 594 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
19 protocols using laser scanning confocal microscope lsm710
1
Assessing Oocyte ROS Levels
2
Cortical Neuron Membrane Potential Imaging
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Cortical neurons were pre-incubated in 5 mM [K+]o solution (130 mM NaCl, 5 mM KCl, 1 mM MgCl2, 15 mM HEPES, 2 mM CaCl2, at 300 mOsm adjusted with glucose) and perfused with 20 or 40 mM [K+]o solution, then washed out by 5 mM [K+]o. Epi-fluorescent imaging experiments were performed with an inverted fluorescence microscope (Ti-U, Nikon, Japan) and Neo sCMOS camera (Andor Technology). The light source was from the mercury lamp filtered at appropriate wavelengths for GFP by the optical filters mounted at the computer-controlled filter wheel (Sutter Instrument) for excitation, subsequently passing the dichroic mirror and the emission filters. Operations and measurements were controlled by the iQ software (Andor Technology). For confocal Ca2+ imaging, a similar protocol was followed with ZEISS Laser Scanning Confocal Microscope (LSM710) (Carl Zeiss) and ZEN 2009 software. Fluorescence intensity (F) was subtracted from its background, to calculate the index of ΔF/F0, where F0 is the baseline fluorescence averaged from 1 s or three or more data points at rest, and ΔF = F−F0.
3
Immunohistochemical Analysis of Hind Paw Skin
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Seventeen weeks after intrathecal inoculation, posterior hind paw skin was isolated and submerged in Zamboni Fixative (NC9335034, Newcomer Supply, Middleton, Wisconsin) overnight and moved to 1x PBS and placed in 4°C for long term storage. Posterior hind paw skins were embedded in O.C.T., sectioned (10 μM, Leica CM3050) and mounted on electrostatically charged slides. For immunohistochemistry, 1–2 slides from 3 mice per treatment group were incubated at room temperature (RT) in 50 mM glycine for 45 minutes and then washed twice, 5 minutes each, in 1x PBST (0.2% triton x-100). Tissues were then blocked for 1 hr at RT in PBST + 10% horse serum (26050–088; Gibco, Thermo Fisher Scientific, Waltham, MA) + 1% bovine serum albumin (BP1600–100, Thermo Fisher Scientific) and then incubated with the primary antibody (ab108986, Abcam, Cambridge, United Kingdom) at 1:500 dilution in blocking solution overnight at 4°C. Tissue was then washed three times, 5 minutes each in PBST and incubated with the secondary antibody (ab150075, Abcam) at a 1:1000 dilution in blocking solution for 4 hr at room temperature. Tissues were washed three more times for 5 minutes each in PBST and then mounted using DAPI Fluoromount-G (0100–20, Southern Biotech, Birmingham, AL). Slides were imaged using a 20x objective on a Zeiss laser scanning confocal microscope (LSM710, Carl Zeiss, Oberkochen, Germany).
4
Immunofluorescent Detection of rFg14-3-3e in Goat PBMCs
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Freshly isolated goat PBMCs were incubated with rFg14-3-3e protein for 1 h in a humidified atmosphere of 5% CO2 at 37 °C. To minimize background staining, goat PBMCs were fixed with 4% paraformaldehyde, washed 3 times in PBS (5 min each) and were treated with blocking solution (4% BSA in PBS) for 1 h at ambient temperature. rFg14-3-3e-treated or non-treated control goat PBMCs were incubated with rat anti-rFg14-3-3e antibody (1:100) for 1 h at 37 °C, followed by staining with Cy3 conjugated goat anti-rat IgG secondary antibody (1:500) (Beyotime, Haimen, Jiangsu, China) for 1 h at 37°C. Hoechst 33342 (Invitrogen, Eugene, Oregon, USA) was used for nuclear staining. Stained cells were imaged with 100× magnification using a Zeiss laser scanning confocal microscope (LSM710, Zeiss, Jena, Germany) and digital images were analyzed by Zen 2012 imaging software.
5
Transfection and Fluorescence Imaging of Cortical Neurons
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2 μg of cDNA encoding jGCaMP7b, jGCaMP7b-XC, or jGCaMP7b-XN and 1 μg cDNA encoding CFP (for labeling the soma area and neurites) were transiently transfected into DIV 5–7 cultured cortical neurons by Lipofectamine 2000 (Invitrogen) with a typical protocol according to the manual. The opti-MEM containing plasmids and Lipofectamine 2000 was added to the Neurobasal medium for transfection. After 2 hr, neurons were maintained in Neurobasal medium supplemented with 2% B27, 1% glutaMAX-I for at least 2 days before analyzing neurite morphology.
Fluorescence imaging of cultured cortical neurons was performed on ZEISS Laser Scanning Confocal Microscope (LSM710, Carl Zeiss) and ZEN 2009 software. N/C ratio of GCaMP or GCaMP-X was calculated by the ratio of fluorescence intensity (nuclear/cytosolic). Measurement of the total length and Sholl analysis for neurites were performed with Imaris 7.7.2 (Bitplane). Only non-overlapping neurons were selected for analysis and images of at least 24 neurons from two independent culture preparations were analyzed. Neurite tracings were depicted with Imaris 7.7.2 in CFP channel.
Fluorescence imaging of cultured cortical neurons was performed on ZEISS Laser Scanning Confocal Microscope (LSM710, Carl Zeiss) and ZEN 2009 software. N/C ratio of GCaMP or GCaMP-X was calculated by the ratio of fluorescence intensity (nuclear/cytosolic). Measurement of the total length and Sholl analysis for neurites were performed with Imaris 7.7.2 (Bitplane). Only non-overlapping neurons were selected for analysis and images of at least 24 neurons from two independent culture preparations were analyzed. Neurite tracings were depicted with Imaris 7.7.2 in CFP channel.
6
Visualizing Autophagy Flux with LC3B Puncta
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To evaluate the formation of fluorescent LC3B puncta, p−mCherry−C1−EGFP−hLC3B (LC3B) was used to monitor autophagy flux, 48 h after LC3B co−transfection with siRNAs, the cells were washed with 1× PBS and immediately analyzed via confocal microscopy (magnification, 500×). The nuclei were counterstained with 4, 6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich). Images were captured using a ZEISS laser scanning confocal microscope (LSM710; Zeiss). ZEISS ZEN Microscope software was used for acquisition.
7
Immunofluorescence Staining of Cultured Cells
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T47D or BT549 cells grown on coverslips were fixed for 15 min with 4% paraformaldehyde in PBS, permeabilized for 10 min in 0.1% Triton X-100 in PBS, and blocked using 5% BSA for 1 h. The cells were then incubated with primary antibodies at 4°C overnight. After a rinse with PBS, the cells were incubated with fluorescent-conjugated secondary antibodies for 1 h at 37°C. The nuclei were counterstained with 4, 6-diamidino-2-phenylindole (DAPI; Sigma–Aldrich). Images were captured using a ZEISS laser scanning confocal microscope (LSM710; Zeiss). ZEISS ZEN Microscope software was used for acquisition.
8
Visualizing Autophagic Flux with mCherry-EGFP-LC3B
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To evaluate the formation of uorescent LC3B puncta, p-mCherry-C1-EGFP-hLC3B (LC3B) was used to monitor autophagy ux, 48 h after LC3B co-transfection with siRNAs, the cells were washed with 1X PBS and immediately analyzed via confocal microscopy (magni cation, x100). The nuclei were counterstained with 4, 6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich). Images were captured using a ZEISS laser scanning confocal microscope (LSM710; Zeiss). ZEISS ZEN Microscope software was used for acquisition.
9
Immunocytochemistry of T47D and BT549 Cells
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T47D or BT549 cells grown on coverslips were xed for 15 min with 4% paraformaldehyde in PBS, permeabilized for 10 min in 0.1% Triton X-100 in PBS, and blocked using 5% BSA for 1 h. The cells were then incubated with primary antibodies at 4°C overnight. After a rinse with PBS, the cells were incubated with uorescent-conjugated secondary antibodies for 1 h at 37°C. The nuclei were counterstained with 4, 6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich). Images were captured using a ZEISS laser scanning confocal microscope (LSM710; Zeiss). ZEISS ZEN Microscope software was used for acquisition.
10
Colonic T-cell Subpopulation Quantification
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