Superoxide production was evaluated in retinal cryosections using dihydroethidium (DHE) as described previously [31 (link)]. Briefly, frozen sections were incubated with DHE (2 μM) for 30 min at 37 °C. DHE is oxidized upon reaction with superoxide to ethidium bromide, which binds to DNA in the nucleus and fluoresces red. Excessive reactive oxygen species (ROS) production is a hallmark of oxidative stress. Thus, retinal levels of superoxide, as determined by DHE staining with subsequent quantification of fluorescence intensity, was measured. Images were captured on a Zeiss LSM 800 confocal scanning microscope. The relative fluorescence intensity within the images obtained was determined via automated image analysis of ZEISS ZEN Intellesis or ImageJ software.
Lsm 800 confocal scanning microscope
Manufactured by Zeiss
Sourced in United States
The LSM 800 is a confocal scanning microscope manufactured by Zeiss. It utilizes laser illumination and a pinhole aperture to capture high-resolution, optical sections of a specimen. The system is designed to provide detailed imaging of samples at the microscopic level.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Rabbit anti calnexin antibody, by Cell Signaling Technology (2 mentions)
Rabbit anti gm130 antibody, by BD (2 mentions)
Alexa fluor 594 488 conjugated goat anti mouse rabbit secondary antibody, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Phosphate buffered saline (pbs), by Merck Group (2 mentions)
Imagej software, by Zeiss (1 mentions)
Alexa 594 goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Alexa 488 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Mouse anti flag antibody, by Merck Group (1 mentions)
Sp8 confocal microscope, by Leica (1 mentions)
Prolong gold antifade, by Thermo Fisher Scientific (1 mentions)
In situ cell death detection kit, by Roche (1 mentions)
Oct compound, by Sakura Finetek (1 mentions)
Cm3050, by Leica (1 mentions)
Superfrost plus microscope slide, by Thermo Fisher Scientific (1 mentions)
Rab11a, by BD (1 mentions)
Donkey anti rat alexa 488, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Goat anti mouse alexa 488, by Thermo Fisher Scientific (1 mentions)
17 protocols using lsm 800 confocal scanning microscope
1
Quantifying Retinal Superoxide via DHE
2
TUNEL Assay for Apoptosis Detection
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Apoptotic cell death was detected by the terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) assay according to the manufacturer’s protocol (Cat#11,684,795,910, Roche Diagnostics, Indianapolis, IN, USA) using prepared frozen sections. Images were captured on a Zeiss LSM 800 confocal scanning microscope.
3
Subcellular Localization of ATP11A
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36-48 hours after COS7 cells transfection, the transfected cells were washed twice with PBS, fixed with 4% paraformaldehyde, and washed three times with PBS. Cells were then permeabilized and blocked with normal goat serum, Triton X-100, and NaN3 in PBS for 1 hour at room temperature. ATP11A protein was labeled with mouse anti-Flag antibody (1 : 2000, Sigma, Germany), and the endoplasmic reticulum (ER) was labeled with a rabbit anti-Calnexin antibody (1 : 1000, Cell Signaling Technology, CA, USA). The Golgi apparatus was labeled with a rabbit anti-GM130 antibody (1 : 1000, BD Biosciences, Mississauga, ON). The secondary antibodies used were Alexa 488 goat anti-rabbit IgG, Alexa 594 goat anti-mouse IgG (1 : 500, Invitrogen, USA). The images were captured under a Zeiss LSM 800 confocal scanning microscope.
4
Immunohistochemistry of Retinal Cone Opsin
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For immunohistochemistry, enucleated eyes were removed, marked at the nasal side for orientation, and fixed for 3 h in 4% paraformaldehyde in 100 mM phosphate buffer (PB) (pH 7.4) and then cryoprotected in 30% sucrose. Tissues were embedded in optimal cutting temperature solution (OCT) and frozen on dry ice for sectioning. Sections (10 micron) were blocked and permeabilized with 10% normal goat serum and 0.1% Triton X-100 in phosphate buffer for 60 min. Labelling with various antibodies was performed as previously described [36 (link)]. Primary antibodies were diluted in phosphate buffer containing 5% normal goat serum and 0.1% Triton X-100. Sections were incubated with primary antibodies overnight. Then, the sections were washed with PB three times and labelled for 1 h with secondary antibody and counterstained with DAPI.
Quantification of mislocalized M-Opsin in the cell bodies of cone cells was performed as follows. P30 and P50 cross-sections of retinas were stained using M-Opsin opsin and DAPI, and higher-magnification images were captured on a Leica SP8 confocal microscope. The number of cones in which M-Opsin mislocalized to the inner segment and to the cell body was counted. Slides were photographed on a Zeiss LSM 800 confocal scanning microscope.
Quantification of mislocalized M-Opsin in the cell bodies of cone cells was performed as follows. P30 and P50 cross-sections of retinas were stained using M-Opsin opsin and DAPI, and higher-magnification images were captured on a Leica SP8 confocal microscope. The number of cones in which M-Opsin mislocalized to the inner segment and to the cell body was counted. Slides were photographed on a Zeiss LSM 800 confocal scanning microscope.
5
Immunohistochemical Staining of Tissue Sections
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Tissues were snap‐frozen in OCT (Sakura) and stored at −80°C. Note that 7 µm sections were cut, air dried for 30 min, fixed in chilled acetone for 20 min, followed by 2× PBS washes. Sections were stained with primary antibodies (Table 1 ) for 20–30 min at room temperature, followed by 2× 5‐min washes in PBS. Secondary antibodies were added for 20–30 min. Slides were washed twice in PBS prior to mounting (ProLong Gold anti‐fade, Thermo Fisher) and imaged on a Zeiss LSM 800 confocal scanning microscope.
6
Immunohistochemistry Protocol for Murine Pancreas
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For immunohistochemistry, mice were anesthetized with a combination of ketamine (16 mg/kg body weight) and xylazine (80 mg/kg body weight) and perfused transcardially with PBS, followed by 4% paraformaldehyde in 100 mM PB (pH 7.4). The dissected tissues were immersed in 4% paraformaldehyde for 2 days at 4°C. Then, the pancreases were removed and dehydrated in 30% sucrose for 24 h. A tissue block containing the pancreas was next prepared and embedded in optimal cutting temperature (OCT) solution and sectioned at a thickness of 10 μm by Leica freezing slicer CM1520. The sections were blocked and permeabilized with 10% normal donkey serum and 0.2% Triton X-100 in PB for 2 h and then incubated overnight at 4°C with the primary antibodies. The primary antibodies were diluted in PBS containing 5% normal donkey serum and 0.2% Triton X-100 and used at concentrations as described in Table S2 . The secondary antibodies were diluted in PBS containing 5% normal donkey serum and 0.2% Triton X-100 at 1:1,000 dilution. TUNEL analysis was performed using an In Situ Cell Death Detection Kit following the manufacturer’s instructions (Roche, Redwood City, CA, USA). The images were captured using a Zeiss LSM 800 confocal scanning microscope. The fluorescence intensity and colocation ratio were measured by Zen software.
7
TUNEL Assay for Apoptosis Detection
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Apoptotic cell death was detected in prepared frozen sections by the terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) assay according to the manufacturer’s protocol (Cat#11684795910, Roche Diagnostics, Indianapolis, IN). Images were captured on a Zeiss LSM 800 confocal scanning microscope.
8
Immunohistochemistry of Mouse Eyes
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For immunohistochemistry, eyes were removed from euthanized mice by intraperitoneal injection of pentobarbital (75 mg/kg) and by cervical dislocation and fixed in 4% paraformaldehyde in 100 mM phosphate buffer (pH 7.4) for 1 h at 4 °C, followed by cryoprotection in 30% sucrose for 2 h. The lenses were removed, and the eyes were embedded in optimal cutting temperature (OCT) solution and sectioned at a 10-μm thickness. After blocking and permeabilization with 10% normal donkey serum and 0.2% Triton X-100 in phosphate buffer for 1 h, the sections were labeled with the primary antibody overnight at 4 °C. The primary antibodies used are shown in Additional file 4 : Table S3. The sections were rinsed in PBS three times, Alexa Fluor 594/488-conjugated goat anti-mouse/rabbit secondary antibody (Cat# A11005 and A11008, Invitrogen, Waltham, MA, USA, 1:500 dilution) was applied, and nuclei were counterstained with DAPI (Cat# D8417, Sigma, St Louis, MO, USA) for 1 h at room temperature. Images were captured on a Zeiss LSM 800 confocal scanning microscope. In addition, the fluorescence intensity was quantified using ZEN 2.3 imaging software.
9
Immunohistochemical Analysis of Mouse Brain
10
Immunofluorescent Analysis of Tight Junctions
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Immunofluorescent staining techniques were used to confirm the presence of tight junctions in cell monolayers. Cells were incubated with 2% paraformaldehyde for 15 min at room temperature, then rendered permeable by exposure to 0.25% Triton X-100 in PBS for 15 min. Monolayers were incubated overnight at 4°C with primary rabbit antibody targeting the tight junction-associated protein zonula occludens-1 (ZO-1), diluted to 1:100 in PBS. A fluorescent-labeled goat anti-rabbit secondary antibody was then applied for 60 min at room temperature, diluted to 1:200 in PBS. Nuclei were counterstained by incubation with the intercalating agent DAPI for 30 min at room temperature, diluted to 1:5000 in distilled water. Transwell inserts were thoroughly washed with PBS between steps. Filter membranes were then excised using a scalpel and mounted onto slides for imaging using a Zeiss LSM 800 confocal scanning microscope.
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