Fluorescence immunocytochemistry was performed on cell cultures in 24-well plates and 35 mm dishes with glass bottoms. The cultures were washed with PBS and then fixed in 4% PFA for 30 min at room temperature. The cells were then washed twice with PBS and nonspecific binding was blocked with 5% normal horse serum, 0.1% Triton X100 in PBS for 1 hr. The cells were subsequently incubated with primary antibodies diluted in blocking buffer overnight at 4°C. For the detailed description of primary antibodies see the section Antibody Characterization below. Cells were subsequently incubated with the appropriate secondary Alexa Fluor 488- or 594-conjugated secondary antibodies (1:500; Gibco). Cell nuclei were counterstained with 4′-6′-diamidino-2-phenylindole (DAPI) (0.5 ug/ml; Sigma). The image areas were randomly selected, avoiding areas within one microscopic field of view from the edge, approximately 400 μm, and areas with either very low cell densities or very clustered nuclei. The immunofluorescence was visualized with an epifluorescence microscope (Zeiss Axiovert LSM510; Carl Zeiss, Göttingen, Germany), and images were obtained on a Macintosh computer using Openlab software (Improvision Inc., Lexington, MA, USA, RRID: rid_000096).
Axiovert lsm510
Manufactured by Zeiss
Sourced in Germany
The Axiovert LSM510 is a confocal laser scanning microscope system designed by Zeiss. It is capable of high-resolution imaging and analysis of biological samples.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Neurolucida 360, by MBF Biosciences (1 mentions)
Neuroexplorer software, by MBF Biosciences (1 mentions)
Cellrox green, by Thermo Fisher Scientific (1 mentions)
Ix71 microscope, by Hamamatsu Photonics (1 mentions)
Lsm image browser, by Zeiss (1 mentions)
Anti mgmt, by Abcam (1 mentions)
9 protocols using axiovert lsm510
1
Fluorescence Immunocytochemistry of Cell Cultures
2
Microglial Morphological Analysis in Hippocampus
3
Quantifying Vector-Derived MeCP2 Expression
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Analysis of expression patterns, transduction efficiency, and quantification of vector-derived MeCP2 levels within nuclei was carried out on image stacks captured using a Zeiss LSM710 or Zeiss Axiovert LSM510 laser confocal microscope (Zeiss). The z series were taken at 1-μm intervals through the section of interest using a 40× objective. To account for antibodies’ penetrability, stack images were taken close to the surface of sections to a maximum depth of 20 μm. To estimate transduction efficiency, images were captured as described earlier, and the ratio of Myc-immunopositive nuclei to DAPI-stained nuclei was calculated for random fields (n = 12 images per region:4 images from each of three mice) from sections of hippocampus (CA1 region), layer 5 of primary motor cortex, thalamus, hypothalamus, brain stem, and striatum. To quantify levels of vector-derived MeCP2 per nucleus in WT mice, confocal stacks (20 μm thick) were obtained as described earlier, and ImageJ software (http://rsbweb.nih.gov/ij/ ) was used to determine mean MeCP2-channel fluorescence intensity within transduced (Myc +ve) and non-transduced (Myc −ve) cells. Fluorescence in the DAPI channel was used to define the nuclear boundary.
4
Quantifying Cellular Oxidative Stress
5
Quantitative Analysis of Neuronal Markers
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Images acquired under a confocal light microscope (Axiovert LSM510; Carl Zeiss Co., Weimar, Germany) were quantitatively analyzed for immunostaining signals using Optical fractionation and MicroBrightField Stereo Investigator software. Total numbers of Nissl+ neurons and TH+ neurons in the SNc, as well as Ki67+ cells and BrdU+ cells in the SVZ, were calculated from six different samples in each group. Before placing the coverslips, a fluorescent mounting medium was applied and allowed to dry in the dark. Specimens were examined with MicroBrightField Stereo Investigator software for counting and photography.
6
Quantifying Neuromuscular Junction Morphology
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NMJ images were acquired on Nikon A1R FLIM and Zeiss Axiovert LSM510 confocal microscopes using established protocols for large volume imaging (Jones et al., 2016 , 2017 ). Muscle fibres were imaged on an Olympus IX71 microscope and Hamamatsu C4742‐95 camera with Openlab Improvision software using the same guidelines (Jones et al, 2016 , 2017 ). For each individual muscle (n = 135), an average of 40–60 NMJs/muscle fibres were imaged, where possible. Muscle fibre diameters were measured subsequently from randomly identified fibres using standard light microscopy (Jones et al., 2016 , 2017 ). It was not possible to record correlated NMJ and muscle fibre measurements from single identified fibres.
Image analysis was performed using the standardized ‘NMJ‐morph’ approach to quantify 21 individual morphological variables in each NMJ (including pre‐ and post‐synaptic variables and associated nerve/muscle measurements; Jones et al,2016 , 2017 and Boehm et al, 2020 ). In total, 5,385 NMJs were analysed across the 6 species, sampled from 135 muscles of 36 individual animals/patients (with mouse and human data pooled from Jones et al., 2017 ).
Image analysis was performed using the standardized ‘NMJ‐morph’ approach to quantify 21 individual morphological variables in each NMJ (including pre‐ and post‐synaptic variables and associated nerve/muscle measurements; Jones et al,
7
Confocal Microscopy of SERT Variants
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Confocal microscopy was carried out as described earlier (28 (link)). HEK293 cells (3 × 105) were seeded onto PDL-coated 15-mm coverslips and transfected with plasmids encoding YFP-tagged wild type and mutant versions of SERT after 24 h using calcium phosphate-mediated precipitation. After an additional 48-h interval, images of the cells (maintained in Krebs-HEPES buffer containing 10 mm HEPES, 120 mm NaCl, 3 mm KCl, 2 mm CaCl2, 2 mm MgCl2, and 2 mm glucose monohydrate, pH adjusted to 7.4 with NaOH) were captured with a Zeiss Axiovert LSM510 confocal laser-scanning microscope (argon laser, 30 milliwatts; helium/neon laser, 1 milliwatt; equipped with an oil immersion objective (Zeiss Plan-NeoFluar ×40/1.3)). The images were analyzed with Zeiss LSM Image Browser (version 4.2.2.121; Carl Zeiss Microimaging GmbH, Oberkochen, Germany).
8
Immunofluorescent Localization of MGMT in Cells
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Cells were plated on glass coverslips 8 hours before treatment. Cells were then fixed with 4% (w/v) paraformaldehyde for 20 minutes at room temperature and permeabilised for 5 minutes in 0.1% Triton X-100/PBS. Cells were blocked for 20 minutes with 5% (w/v) gelatin/PBS in a humidified chamber. Cells were incubated with anti-MGMT (Abcam, UK) for 1 hour at room temperature also in a humidified chamber. Alexa Fluor 555-conjugated anti-rabbit secondary antibody was added for 1 hour at room temperature. Alexa Fluor 488 phalloidin was also used in the secondary antibody incubation. Following further extensive washing, nuclei were stained with DAPI (4’, 6-diamidino-2-phenylindole) for 5 minutes. After a further two washes, coverslips were mounted on microscope slides with Mowiol mounting solution (0.2 M Tris/HCl (pH 8.5), 33% (w/v) glycerol, 13% (w/v) Mowiol and 2.5% (w/v) DABCO (1, 4-diazobicyol [2 (link)]-octane)) and sealed with clear nail polish. Images were collected on a Zeiss Axiovert LSM510 scanning confocal microscope using a × 100 objective. Single stain, bleed-though controls and antibody cross-reaction controls were prepared for each sample (results not shown).
9
PKCε Mobilization in PC-12 Cells
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PKCε, conjugated with the red fluorescent protein eqFP611, mRuby, was expressed into PC-12 cells, and intracellular PKCε mobilization was monitored with a confocal scanning laser microscope (Axiovert/LSM510; Carl Zeiss, Oberkochen, Germany).
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