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8 protocols using fluoview 1000 confocal laser scanning system

1

Analyzing Mitophagy in C2C12 Cells

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Cells were seeded directly on glass coverslips and after 48 h were fixed with 4% paraformaldehyde and permeabilized by incubation with 0.2% Triton X-100. For mitophagy analysis, C2C12 cells were co-transfected with scramble (scr) or PGC-1α siRNA (PGC-1α(−)) and EGFP-LC3 and incubated with mouse HSPA9 (1 : 50) antibody. After staining with the appropriate AlexaFluor-conjugated secondary antibodies (1 : 1000), cells were incubated with Hoechst 33342 (Life Technologies, Carlsbad, CA, USA) to visualize nuclei. Images were visualized with an Olympus Fluoview 1000 confocal laser scanning system (Segrate, Milano, Italy). The images presented were captured under constant exposure time, gain and offset. Pearson's correlation coefficient, R(r), was calculated for fluorescence intensities of both the confocal channels. This coefficient describes the correlation between the intensity distribution or pattern overlap in two channels in terms of a least squares fit. This value can be between −1 and 1, and R=1 indicates complete correlation between the two channels. Finally, the overlap coefficient indicates an overlap of the signals and thus represents the true degree of colocalization.
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2

Immunofluorescence Assay for DRG2 in Tissue Sections

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Immunofluorescence assays for DRG2 were performed on paraffin-embedded muscle, heart, and liver tissue sections. The sections were incubated in different concentrations of ethanol and washed in PBS. The sections were boiled in an antigen retrieval buffer (10 mM sodium citrate buffer, pH 6.0, 0.5 ml Tween 20) for 10 min and washed in PBS. The sections were incubated in 0.3% H2O2 for 10 min at RT and blocked with 5% Normal Goat Serum for 1.5 h at RT. The sections then were incubated with primary antibodies against DRG2 (1 : 250 dilution in 5% Normal Goat Serum) overnight at 4°C, followed by the fluorescence-labeled secondary antibody Alexa Fluor 568 goat anti-rabbit (1 : 1,000). Nuclei were stained with DAPI (1 : 2,000) for 5 min at RT. The sections were mounted on glass slides and viewed on an Olympus FluoView 1000 confocal laser scanning system.
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3

Histological and Immunofluorescence Analysis

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For histological analysis, tissues were fixed in 10% formalin solution, embedded in paraffin, and sectioned. H&E staining were performed as described previously [22 ].
For immunofluorescence (IF) staining of tissue and primary adipocytes, snap-frozen tissues were sectioned using a cryostat and then defrosted at room temperature for 1 h. The primary adipocytes and tissue sections were fixed in 4% paraformaldehyde (HT5014; Sigma-Aldrich) at room temperature for 20 min, washed thrice in PBS, incubated with 0.2% Triton X-100 for 5 min, and rinsed in PBS. The sections were blocked with 5% goat serum with 0.1% Triton X-100, whereas adipocytes were blocked with 3% bovine serum albumin (BSA) for 1 h at room temperature. Then, both were incubated with the target protein antibody (1:200) with their respective blocking solution overnight at 4 °C. The next day, the tissues and adipocytes were again washed thrice with PBS, stained with Alexa Fluor-conjugated secondary antibody in blocking solution at room temperature in the dark for 1 h, washed twice with PBS, and incubated with 10 μM DAPI in PBS at room temperature for 10 min. Images were acquired and analyzed using an Olympus FluoView 1000 confocal laser scanning system. The slides were scored in a blinded manner.
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4

Visualizing Mitochondrial Morphology in Cells Expressing DJ-1 Variants

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Cells transiently transfected with wt and mutant DJ-1 were cultured on poly-D-lysine glass coverslips (24Ø) for 24 h. After administration of CCCP 10 μM for 8 h, cells were washed twice in cold PBS (pH 7.4) and fixed in 4% (w/v) paraformaldehyde for 20 min at RT. Following three washes in PBS, cells were permeabilized with 0.1% Triton-X100 (AppliChem PanReac) for 10 min at RT and blocked with 3% (w/v) bovine serum albumin for 1 h at RT. All coverslips were then incubated with primary antibodies (goat polyclonal anti-DJBP at 0.4 μg/ml, Santa Cruz) and rabbit polyclonal anti-ATPβ at 1 μg/ml (Abcam) at 4°C overnight. Following three washes in PBS, coverslips were incubated in the dark with a mix of the following secondary antibodies AlexaFluor 594-conjugated anti-goat IgG and AlexaFluor 488-conjugated anti-rabbit IgG (Invitrogen) at 1:100 for 1 h at RT. After a final wash, coverslips were mounted with ProLong Gold antifade reagent (Invitrogen) containing DAPI (Applichem), for nuclei visualization, and imaged by means of an Olympus Fluoview 1000 Confocal Laser Scanning System. All images were analyzed by ImageJ software (NIH, Bethesda, MD, United States).
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5

Confocal Microscopy Analysis of Photosynthetic Pigments

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Microscopy analyses were performed using a confocal laser scanning microscope coupled with spectral analysis (CLSM-λscan). Small fragments (about 2 mm2) were put onto slides and examined using a CLSM (Olympus Fluoview 1000 Confocal Laser Scanning System). Autofluorescence of photosynthetic pigments (chlorophyll a and phycobiliproteins) and minerals was investigated by successively exciting the samples with 488-nm, 543-nm and 635-nm lasers, and collecting the emitted fluorescence in three channels: 503–524 nm, 555–609 nm and 655–755 nm. Three-dimensional images were captured every 0.5 μm and processed with Imaris v. 6.1.0 software (Bitplane AG Zürich, Switzerland) to obtain maximum intensity projections. The spectral analysis of regions of interest (ROI) was performed using the 543-nm laser at 54 % of the maximum power (=0.54 mW) and collecting the emission from 553 to 800 nm, and mean fluorescence intensity (MFI) was measured. Curve plotting was performed using the GraphPad Prism program (GraphPad Software, San Diego, CA).
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6

Immunofluorescence Analysis of DNA Damage

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Cells were grown on glass coverslips and treated with H2O2 or transfected with plasmid for 72 h. Cells were washed with PBS and fixed with 4% paraformaldehyde for 20 min at room temperature (RT). After washing with PBS twice, the cells were permeabilized with ice cold 0.2% Triton X-100 (T8787; Sigma-Aldrich) for 5 min and then blocked with 3% Normal Goat Serum (#31872; Thermo Fisher) for 30 min at RT. Then, the cells were incubated with anti-DRG2 (1 : 250, rabbit; Proteintech), anti-SIRT1 (1 : 250, mouse; Abcam), and p-H2A.X (1 : 250, rabbit; CST) overnight at 4°C, washed twice with PBS, and incubated with Alexa Fluor 568 goat anti-rabbit antibody (1 : 1,000), Alexa Fluor 488 goat anti-mouse antibody (1 : 1,000), and Alexa Fluor 568 goat anti-mouse antibody (1 : 1,000) for 30 min at RT, respectively. The cells were stained with 4′6-diamidino-2-phenylindole (DAPI) (P36931; Sigma-Aldrich), washed with PBS twice, mounted on glass slides, and viewed on an Olympus FluoView 1000 confocal laser scanning system (Olympus, Tokyo, Japan).
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7

Biofilm Viability Assessment by Microscopy and Staining

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Cells from dried biofilms and dried-UV-irradiated biofilms were observed with a CLSM (Olympus Fluoview 1,000 Confocal Laser Scanning System). Images were taken using a 60× objective and photosynthetic pigment autofluorescence was investigated by exciting the cells with a 543- and a 635-nm laser and collecting the emission from 645-nm, or from 553-nm, to 800-nm emission range.
Viability was assessed: (1) by inoculating biofilm fragments (about 25 mm2) into 2 ml of liquid BG-11 medium and measuring cell densities with a spectrophotometer after 3 months of growth under routine conditions and (2) by staining with 2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-phenyl tetrazolium chloride (Sigma Aldrich, Saint Louis, MO, USA) after rehydration for 30 min, 60 min, and 72 h, as previously reported (Billi, 2009 (link)).
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8

Confocal Microscopy for Mitochondrial Analysis

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Olympus Fluoview 1000 confocal laser scanning system was used for colocalization experiments. The 488 nm laser was used to excite MTG and AlexaFluor 488-conjugated secondary antibody (Life Technologies). The 543 nm laser was used for detection of MTR and AlexaFluor 568 conjugated secondary antibody (Life Technologies). A 63X objective was used for all images. Nuclei were stained with Hoechst 33342 (10 μg/ml). Mitochondrial morphology analysis was performed by using Mitophagy Macro Plugin (ImageJ Software), and mitochondria area, perimeter and circularity were reported.
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