Images were captured on a Zeiss LSM710 confocal microscope and processed with Zen software, ImageJ/Fiji, and Adobe Photoshop. For imaging based quantification, unless otherwise specified, 3 representative images from different mice were quantified manually and data were plotted with Graphpad.
Lsm710 confocal microscope
Manufactured by Adobe
Sourced in United States
The LSM710 is a confocal microscope that uses laser excitation to capture high-resolution, three-dimensional images of samples. It provides a core function of enabling detailed imaging and analysis of microscopic specimens.
Automatically generated - may contain errors
Lab products found in correlation
Vectashield, by Vector Laboratories (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (3 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (2 mentions)
Vectorshield, by Vector Laboratories (2 mentions)
Imagej, by Adobe (1 mentions)
Alexa fluor 488 goat anti rabbit igg, by Cell Signaling Technology (1 mentions)
Apc 107, by Alomone (1 mentions)
Cm h2dcfda reagent, by Thermo Fisher Scientific (1 mentions)
Fluoromount g mounting medium, by Southern Biotech (1 mentions)
A10262, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Eclipse ti e microscope, by Nikon (1 mentions)
Rabbit anti gfp, by Thermo Fisher Scientific (1 mentions)
Mouse anti mira, by Merck Group (1 mentions)
Rat anti cd8, by Thermo Fisher Scientific (1 mentions)
Rabbit anti h4k20me1, by Merck Group (1 mentions)
Fitc conjugated anti hrp, by Jackson ImmunoResearch (1 mentions)
Anti brp, by Developmental Studies Hybridoma Bank (1 mentions)
Alexa 488 and 568, by Thermo Fisher Scientific (1 mentions)
Texas red x phalloidin, by Thermo Fisher Scientific (1 mentions)
17 protocols using lsm710 confocal microscope
1
Confocal Microscopy Imaging Protocol
2
Immunostaining of Drosophila Larval Tissues
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Briefly, third instar larvae were dissected for larval brains, imaginal discs, and gut in 1xPBS and fixed in 3.7% formaldehyde for 30 min at room temperature and washed in 0.2% Triton X-100 (Nagarkar-Jaiswal et al., 2015 (link)). They were then incubated for 1 hr at RT in 10% NGS-PBS-0.2% Triton X-100 and stained with primary antibodies diluted in 10% NGS-PBS-0.2% Triton X-100 overnight at 4°C. The samples were washed and incubated with secondary antibodies for 2 hr at RT. The samples were then washed and mounted in Vectashield (Vector Labs, Burlingame, CA) and imaged with a Zeiss LSM710 confocal microscope and processed using Adobe Photoshop (Adobe Systems Inc., San Jose, CA, USA).
3
Immunofluorescence Staining of Podocyte KCa1.1
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Podocytes were fixed in 4% paraformaldehyde in PBS for 15 min at room temperature. After rinsing twice with PBS, cells were blocked with 2% bovine serum albumen (BSA) in PBS for 1 h at room temperature. Cells on coverglass were incubated with KCa1.1 (1184–1200) antibody (APC-107, Alomone Labs) at a 1:100 dilution in PBS with 2% BSA at 4°C overnight. Cells were rinsed three times with PBS containing 0.02% Tween 20. Secondary antibodies were diluted in PBS with 2% BSA and cells were incubated at room temperature for 1 h. Alexa Fluor 488 goat anti-rabbit IgG (1:500; Cell Signaling Technology, United States) served as the secondary antibody. After rinsing three times with PBS containing 0.02% Tween 20, cells were incubated with DAPI (2 μg/ml) for 1 min. Cells were rinsed three times with PBS containing 0.02% Tween 20 and cells on coverglass were mounted on microscope slides with Prolong Gold antifade reagent (Invitrogen, United States). Images were taken using a Carl Zeiss LSM710 confocal microscope and processed using Photoshop software (Adobe Systems, Inc., San Jose, CA, United States). All of the images were quantified with ImageJ software and normalized to the respective control.
4
Extracellular ROS Scavenging by CeNP-PEG
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HK-2 cells were treated with100 μM of H2O2 for 2 h with the presence of the CeNP-PEG at different concentrations (0, 0.25, 0.50 and 1.00 μg/mL) or vehicle. The effect of CeNP-PEG on extracellular ROS scavenging was investigated by DCFH-DA. Briefly, cells were loaded with CM-H2-DCFDA reagent (Invitrogen) by incubating cells with 5 μM of probe solution in PBS for 20 min at 37 °C. Then, the fluorescence microscopy was visualized using a Carl Zeiss LSM710 confocal microscope, and processed using Photoshop software (Adobe Systems, Inc., San Jose, CA, USA).
5
Immunostaining of Adult Fly Brains
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Immunostaining of whole-mount adult fly brains was performed as previously described50 (link). Dissection of adult brains was done in ice-cold PBS and fixed in 4% paraformaldehyde (PFA) prepared in PBS for 20 min. Brains were mounted in Fluoromount-G mounting medium (Southern Biotechnology, 0100–01). Primary antibodies used were anti-Ringer (1:750)10 (link), anti-Elav (1:500, DSHB, 9F8A9), anti-Brp (1:250, DSHB, NC82), anti-Th (1:300, Novus Biologicals, NB300-109), anti-GFP (1:500, Invitrogen, A10262), and anti-Repo51 (link). Secondary antibodies conjugated to Alexa 488, 568 and 647 (Invitrogen-Molecular Probes) were used at 1:400 dilution. Confocal images of all genotypes of the brains belonging to the same experimental group were acquired using the same settings with a Zeiss LSM710 confocal microscope, and image editing was done using Adobe Photoshop software.
6
Confocal Microscopy Image Analysis
7
Immunofluorescence Staining of HK-2 Cells
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HK-2 cells cultured on cover slips were fixed in 4% formaldehyde for 15 min, permeabilized in 0.25% Triton X-100 for 10 min, and blocked in quick blocking liquid for one hour at room temperature. Then, the primary antibody for α-SMA (1:200) or Vimentin or FN (1:100) was incubated overnight at 4 °C. Next, cells were rinsed for three times with PBS containing 0.02% Tween 20. Then, the secondary Alexa Fluor 488 goat anti-rabbit IgG (CST, USA) was incubated at a dilution of 1:200 for one hour at room temperature. After rinsing for three times with PBS containing 0.02% Tween 20, these cells were incubated with DAPI (2 mg/ml) for 1 min. Then, these cells were rinsed for three times with PBS containing 0.02% Tween 20, and cells on the cover glass were mounted on microscope slides with the Prolong Gold anti-fade reagent (Invitrogen, USA). Images were taken using a Carl Zeiss LSM710 confocal microscope, and processed using the Photoshop software (Adobe Systems Inc., San Jose, CA, USA).
8
Drosophila Ovary and Larval Tissue Staining
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Adult Drosophila ovaries were dissected and stained as described in ref. 79 (link). Wandering third instar larval tissues were processed similarly to adult ovaries with one additional permeabilization step with 0.1% TritonX-100 in 1X PBS for 1 h at room temperature immediately following fixation. Primary antibodies were diluted in 5% BSA, 0.3% TritonX-100 in PBS and are listed in Supplementary Table 2 . Alexa Fluor-conjugated secondary antibodies were used at a 1:500 dilution. All images were collected on a Zeiss LSM 710 Confocal Microscope, cropped and rotated using ImageJ software, size matched with Adobe Photoshop and assembled with Adobe Illustrator. The number of HP1a foci was quantified manually using ImageJ. Unless otherwise noted, all images are a single slice of a confocal stack.
9
Immunofluorescence Imaging of WNV-Infected Vero Cells
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Vero cell monolayers on coverslips were infected with WNVKUN and incubated at 37°C for 24h. The cells were subsequently washed with PBS and fixed with 4% paraformaldehyde (Sigma Aldrich, St. Louis, Mo.) and permeabilised with 0.1% Triton X-100 as previously described (20). Primary and secondary antibodies were incubated within blocking buffer (PBS containing 1% BSA) and washed with PBS containing 0.1% BSA between incubation steps. After a final wash with PBS the coverslips were drained and mounted onto glass slides with a quick dry mounting medium (United Biosciences, Brisbane, Australia). Images were collected using a Zeiss LSM710 confocal microscope and Zen software before processing for publication using Adobe Photoshop software.
10
Immunohistochemistry for Glyoxal/PFA-Fixed Brain
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Immunohistochemistry was performed as described in our previous publications.56 (link) Briefly, brain sections were fixed in cold 3% glyoxal for 30 min or 4% PFA for 20 min. After extensive washes with PBS, the sections were incubated in blocking buffer (1% BSA in PBS containing 0.3% normal donkey serum and 0.3% Triton X-100) for 1 h at room temperature. Next, the sections were incubated with primary antibodies overnight at 4°C. After extensive washes in PBS, the sections were incubated with appropriate secondary antibodies for 1 h at room temperature. Then, the sections were washed in PBS for 3 times and mounted with Fluoromount-G with DAPI. Nikon Eclipse TiE microscope and LSM710 confocal microscope were used to take images, which were further processed by ImageJ and/or Adobe Photoshop. For ICH brains, images were taken from the peri-hematoma regions.
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