Cells stably transduced with wild-type or mutated mitoStat3 and non-transduced cells were seeded in 60 × 15 mm culture dishes (20 × 103 cells/cm2) in complete fresh medium, incubated for 24 h and then irradiated with UVC. The activation of caspases 3 and 9 was measured by the Caspase-9 Fluorimetric assay kit (BioVision, Abcam, Waltham, MA, USA) at 24 h after irradiation. The fluorescent emission at λ = 505 nm (excitation at λ = 400 nm) of cleaved amino-4-trifluoromethyl coumarin (AFC) was measured in a 96-well plate reader (EnVision, Perkin Helmer). Apoptotic cells were recognized based on the fragmented morphology of their nuclei stained with 4,6-diamino-2-phenylindol (DAPI, Roche, Basel, Switzerland, 2 mg/mL), following standard protocol [54 (link)]. At least 500 cells/sample/experiment were scored under a LeicaDM4000 fluorescent microscope (Leica Microsystems, Wetzlar, Germany).
Dm4000 fluorescent microscope
Manufactured by Leica
Sourced in Germany
The Leica DM4000 is a fluorescent microscope designed for advanced imaging applications. It features a high-performance optical system and a range of advanced features to support a variety of research and analytical tasks. The DM4000 is capable of delivering high-quality, detailed images of fluorescently labeled samples.
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Lab products found in correlation
Gl7 fitc, by BD (2 mentions)
Caspase 9 fluorimetric assay kit, by Abcam (1 mentions)
Everbrite mounting medium, by Biotium (1 mentions)
Fluorescein isothiocyanate fitc labelled phalloidin, by Merck Group (1 mentions)
Alexa fluor 488 conjugated goat anti rat antibody, by Thermo Fisher Scientific (1 mentions)
Aqua poly mount medium, by Polysciences (1 mentions)
Alexa fluor 555 conjugated streptavidin, by Thermo Fisher Scientific (1 mentions)
Biotin blocking system, by Agilent Technologies (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Goat anti mouse alexa fluor 546, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Tcs sp5 confocal microscope, by Leica (1 mentions)
12 protocols using dm4000 fluorescent microscope
1
Assessing Mitochondrial Stat3-Induced Apoptosis
2
Fluorescent Labeling of FB Samples
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The samples of FB1 –FB4 were incubated for 18 h at 4 °C in PBS containing green fluorescent protein (GFP; Evrogen, Moscow, Russia) with a concentration of 0.2 mg/mL, washed three times by PBS and studied using a Leica DM 4000 fluorescent microscope (Leica Microsystems GmbH, Wetzlar, Germany).
3
Immunofluorescence Staining of Podocytes
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Human immortalized podocytes were grown on poly-D-lysine-four-well chamber slides to 50–60% confluence. After removing the medium, cells were washed twice thoroughly with PBS followed by fixation with 4% freshly prepared paraformaldehyde and blocking with 5% donkey serum for 20 minutes. The cells were stained with primary anti-synaptopodin (sc-21537, 1 : 200, Santa Cruz Biotechnology Inc., Heidelberg, Germany) and secondary Cy3-conjugated antibodies. For the visualization of the cytoskeleton, fluorescein isothiocyanate- (FITC-) labelled phalloidin (Sigma-Aldrich, St. Louis, MO, USA) at a dose of 50 μg/mL for 40 minutes was applied to stain F-actin. To optimize detection, streptavidin/biotinylated horse-radish peroxidase system was applied for amplification of the signal. Finally, the samples were covered with a DAPI (4′,6-diamidino-2-phenylindole) containing antifade mounting medium (EverBrite mounting medium, Biotium Inc., Hayward, CA, USA) to stain the nuclei and minimize photobleaching of the samples. Pictures were taken using a Leica DM4000 fluorescent microscope equipped with a Leica DFC310 FX digital color camera (Leica Microsystems GmbH, Wetzlar, Germany).
4
Immunofluorescent Staining of Mouse Kidney Cryosections
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Cryosections from snap-frozen mouse kidneys were fixed in acetone for 10 min at room temperature. Sections were encircled with a wax pen and rehydrated in PBS. Endogenous biotin was blocked with a Biotin Blocking system (Dako). Primary antibodies against Axl, Fyn, or CD31 (Table 1 ) were diluted in 5% (v/v) FCS in PBS and sections were incubated for 1 h at room temperature. After washing in PBS, sections were incubated with either AlexaFluor®555-conjugated rabbit anti-goat antibody (Thermo Fisher Scientific), AlexaFluor®488-conjugated goat anti-rat antibody (Thermo Fisher Scientific) to detect Axl, or biotin-conjugated goat anti-rabbit antibody (Southern Biotech, Uden, Netherlands) to detect Fyn, followed by AlexaFluor®555-conjugated streptavidin (Thermo Fisher Scientific) to stain CD31. All secondary antibodies were diluted in 5% FCS/PBS. After incubation, sections were washed in PBS and mounted in Aqua-Poly/Mount medium (Polysciences, Hirschberg an der Bergstrasse, Germany) containing 4′, 6-diamidino-2-phenylindole (Invitrogen, Leiden, Netherlands). Fluorescent images were taken with equal exposure times using a Leica DM4000 fluorescent microscope (Leica Microsystems, Germany) with Leica LAS V4.5 image software.
5
Gamma Irradiation-Induced DNA Damage Analysis
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Stable cell lines, expressing KIN or GFP-NLS, were irradiated using gamma rays at 10 grays and harvested 2 or 24 h later for BioID purification; non-irradiated cells were used as control. For DNA damage identification, cells were cultivated on coverslips at the bottom of the plate, and underwent the same irradiation treatment. Cells were fixed in freshly prepared 4% paraformaldehyde in PBS, permeabilized in 0.5% Triton X-100 in PBS, blocked in 2.5% goat serum, 1% BSA. Cells were incubated in anti-H2AX (monoclonal #05-636, Millipore) 1:5000, and goat-anti-mouse Alexa Fluor 546 (Thermo Fisher Scientific, Waltham, MA, USA) 1:500. Nuclear staining was performed using DAPI (Sigma, Burlington, VT, USA), and coverslips were mounted on slides using one drop of mounting medium (Aqua-Mount #13800, Thermo Fisher Scientific). Images were acquired using the DM4000 fluorescent microscope (Leica, Wetzlar, Germany). One replicate of KIN and one replicate of GFP-NLS were used for each point, non-irradiated, irradiated and harvested after two hours, and irradiated and harvested 24 h later (Figure S2 ).
6
HEp-2 Indirect Immunofluorescence Assay
7
Quantification of DNA Damage and Cell Fate
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For DSB (cells and slices) and micronuclei quantification, Z-stack imaging was performed using a TCS SP5 confocal microscope (Leica) and foci were counted from at least 50 cells per condition per experiment (cells) or on average 650 cells per condition per slice (slices) using Image J software (settings: median blur 1.0, maximum projection and find maxima, noise tolerance 75 for cells and 100 for slices). Micronuclei of least 50 cells per condition per experiment were counted manually and expressed as fraction of cells with micronuclei. For cytochrome C release quantification, imaging was performed using a DM4000 fluorescent microscope (Leica). At least 50 cells per condition per experiment were counted manually and expressed as fraction of cells with released cytochrome C (cytoplasmic and total release). For EdU staining, imaging was performed using a DM4000 fluorescent microscope and at least 100 cells per condition per experiment were analyzed. H&E stainings are image using the Olympus BX40 phase contrast microscope. Quantification data is presented as mean with standard error.
8
DNA Replication Progression Assay
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DNA replication progression was analyzed using the DNA fiber assay (Xu et al., 2011 (link); Liu et al., 2016 (link)). Cells were labeled with IdU (25 μM) for 30 min, followed by exposure to HU (4 mM) or DMSO for 4 h, and chased with CldU (125 μM) for 40 min. Cells were lysed, DNA fibers were spread, and the IdU and CldU tracts were stained and detected, as previously described (Xu et al., 2011 (link); Liu et al., 2016 (link)). DNA fibers were imaged using a Leica DM4000 fluorescent microscope.
9
Visualization and Quantification of Germinal Centers
10
Imaging Native Chromomeres on Lampbrush Chromosomes
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To obtain pictures of native chromomeres, freshly prepared lampbrush chromosomes were stained with 1 μg/ml DAPI in antifade solution described above and imaged with Leica DM4000 fluorescent microscope before FISH. 2D-FISH procedures were applied to lampbrush chromosome preparations according to DNA/DNA+RNA protocol preserving RNA (Solovei et al. 1994; (link)Zlotina and Krasikova 2017) . In particular, RNAase treatment was omitted.
Chromosomes and DNA-probes were denatured separately as described for 3D-FISH. 1-5 μl drops of denatured hybridization mix were applied to the slides with denatured lampbrush chromosomes, mounted with coverslip and sealed with rubber cement. Hybridization was carried out overnight at 37°C in a humid chamber. Post-hybridization washing was performed at 60°С in (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint this version posted November 30, 2021. ; https://doi.org/10.1101/2021.11.30.470320 doi: bioRxiv preprint 0.2×SSC. Biotin and digoxigenin were detected as described above. Slides were mounted with the antifade solution containing DAPI.
Chromosomes and DNA-probes were denatured separately as described for 3D-FISH. 1-5 μl drops of denatured hybridization mix were applied to the slides with denatured lampbrush chromosomes, mounted with coverslip and sealed with rubber cement. Hybridization was carried out overnight at 37°C in a humid chamber. Post-hybridization washing was performed at 60°С in (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint this version posted November 30, 2021. ; https://doi.org/10.1101/2021.11.30.470320 doi: bioRxiv preprint 0.2×SSC. Biotin and digoxigenin were detected as described above. Slides were mounted with the antifade solution containing DAPI.
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