PAM were grown on coverslips and treated or untreated with cGAMP (20 μg/ml) or mock or ASFV infected at the indicated MOI. At the indicated times postinfection (1 or 6 hpi) or 1 h after cGAMP treatment, the cells were fixed with 4% paraformaldehyde for 20 min at room temperature, permeabilized with 0.2% Triton X-100 for 15 min, and blocked with PBS–5% BSA for 45 min. The cells were stained with the primary antibodies anti-STING antibody (1/50) and anti-γ-adaptin (AP1) antibody (1/100) diluted in PBS–1% BSA for 1 h. The cells were then washed with PBS and incubated with the fluorescence-conjugated secondary antibodies anti-rabbit/Alexa Fluor 488 (1/500) and anti-mouse/Alexa Fluor 555 (1/500) diluted in PBS–1% BSA for 1 h. After a wash with PBS, the coverslips were mounted with DAPI Fluoromount-G (SouthernBiotech). Images were acquired by using a CLSM LSM710 coupled to an inverted AxioObserver microscope (Zeiss) with a 63× oil immersion objective lens and analyzed using ImageJ. Colocalization between STING and AP1 was quantified by calculating Mander’s coefficient (M1) using the plugin JACoP (Just Another Colocalization Plugin; Fiji).
Clsm lsm710
Manufactured by Zeiss
Sourced in Germany
The CLSM LSM710 is a confocal laser scanning microscope (CLSM) developed by Zeiss. It is a high-resolution imaging system designed for biological and materials science applications. The CLSM LSM710 uses a focused laser beam to scan the sample, and the reflected or fluorescent light is detected and processed to generate a detailed, three-dimensional image of the specimen.
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Lab products found in correlation
Leptomycin b lmb, by Cell Signaling Technology (2 mentions)
Hoechst 33258, by Thermo Fisher Scientific (2 mentions)
Inverted axioobserver microscope, by Zeiss (1 mentions)
Dapi fluoromount g, by Southern Biotech (1 mentions)
Countess cell counting chamber slide, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 647 labeled dextran conjugate, by Thermo Fisher Scientific (1 mentions)
Syto9, by Thermo Fisher Scientific (1 mentions)
Alexa flour 488 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Phospho stat3 antibody, by Cell Signaling Technology (1 mentions)
Live dead baclight bacterial viability kit, by Thermo Fisher Scientific (1 mentions)
Plan apochromat 63 1.4 objective, by Zeiss (1 mentions)
8 protocols using clsm lsm710
1
STING-AP1 Colocalization Analysis
2
Microfluidic Droplet Encapsulation and Protein Quantification
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Single emulsion droplets were loaded into Hollow Rectangle Capillaries ID 0.05 x 0.50mm (CM Scientific) or into Countess Cell Counting Chamber Slides (ThermoFisher Scientific) for microscopy imaging. Images were taken on a CLSM LSM710 (Zeiss) upright confocal microscope with 10X objective. Parallel images were taken with transmitted light and the corresponding fluorescence laser and filter setting for mNeonGreen (excitation 488 nm, emission 515–564 nm) or LSSmOrange (excitation 440 nm, emission 536–706 nm) or mScarlet-I (excitation 561 nm, emission 589–740 nm). A droplet-based protein concentration calibration curve was prepared from purified mNeonGreen protein. Four different concentrations of mNeonGreen were encapsulated with the μEncapsulator system and the fluorescence was measured under identical conditions (S2 Fig in S1 File ). Images were analyzed in MATLAB with a script modified from a script kindly provided by Daniela Garcia (UPF, Barcelona, Spain) ([https://github.com/strubelab/dropletXpress ]) and protein concentrations were determined from the median fluorescence intensity.
3
Visualizing MUC1-C Localization in SARS-CoV-2 Infected Cells
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MUC1-C expression and localization was confirmed by confocal imaging as described previously (35 (link), 36 (link)). Briefly, Calu-3 cells were cultured for 24 h on poly-L-lysine-coated glass cover slips in 12-well culture plates. The cells were washed with PBS and infected with SARS-CoV-2 at an MOI of 0.5. After 1 h, viral supernatants were removed and replaced with 2 ml of DMEM containing 2% FBS. The cells were incubated for 45 h at 37°C in a CO2 incubator prior to treatment with PBS or Leptomycin B (LMB, Cell Signaling Technology, 20 nM), an inhibitor of chromosomal region maintenance 1 (CRM1) that is a nuclear export protein. After a 3 h treatment, the cells were prepared for immunofluorescent imaging as follows. Cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, blocked with 3% BSA, and incubated with rabbit anti-MUC1-CT antibody for 2 h at room temperature. After washing the cells with PBST (0.1% Triton X-100 in PBS) containing 1% BSA, the cells were incubated with Alexa Fluor 488-conjugated secondary antibody (Catalog No. A32790, Thermo Fisher Scientific) for 1 h at room temperature. Nuclei were stained with Hoechst 33258 (Thermo Fisher Scientific). Images were collected using a confocal laser scanning microscope system (CLSM, LSM 710, Carl Zeiss).
4
Visualizing BPA Uptake in BxPC-3 Cells
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BxPC-3 cells were seeded on a glass-base dish (5 × 104 cells per dish) and incubated for 24 hours. After removing the medium, the cells were incubated in medium [D-PBS/RPMI = 1/4 (v/v)] containing fructose-BPA (fructose: 290 μM, BPA: 110 μM) or Cy5-PVA-BPA (diol unit in Cy5-PVA: 340 μM, BPA: 110 μM) for 30 min. The cells were then incubated in D-PBS containing 20 μM DAHMI and 50 nM LysoTracker Red DND-99 for 30 min, followed by washing with D-PBS thrice. The cells in D-PBS were observed using CLSM (LSM710, Carl Zeiss AG, Oberkochen, Germany).
5
Streptococcus mutans Biofilm Characterization
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Streptococcus mutans UA159 strains grown to mid-logarithmic phase (OD600 = 0.5) were diluted 1:100 in BHI containing 1% sucrose. For biofilm formation, aliquots (3 ml) of S. mutans strains in BHI-1% sucrose were placed into a 24-well plate containing polystyrene blocks and cultured at 37°C anaerobically for 24 h allowing biofilm formation. To determine the biomass and analyze structure alteration, SYTO 9 (Molecular Probes, Invitrogen, Carlsbad, CA, United States) was employed to label bacterial cells. As the main component of biofilm, exopolysaccharides were labeled with Alexa Fluor 647-labeled dextran conjugate (Molecular Probes) as previously described (Koo et al., 2010 (link)). Images were obtained with CLSM (LSM 710, Carl Zeiss, Germany) with a 20× objective and image series were generated by optical sectioning at each position. The three dimensional re-construction of biofilms was performed and the biomass of EPS and bacteria was analyzed using COMSTAT software.
6
SARS-CoV-2 Induces STAT3 Phosphorylation
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Induction of STAT3 phosphorylation and phospho-STAT3 nuclear localization by SARS-CoV-2 were observed by indirect immunofluorescence and confocal microscopy. Calu-3 cells were cultured on glass coverslips in 12-well plates overnight. The cells were pretreated with DMSO, 25 μM AG490, 1 μM JAK inhibitor I, 20 μM S3I-201, or 10 μM STA-21 for 30 min and then infected with SARS-CoV-2 in PBS (0.5 MOI) for 1 h at 37°C. The cells were cultured in DMEM containing 2% FBS for 48 h and then treated with Leptomycin B (LMB, Cell Signaling Technology, 20 nM), an inhibitor of the nuclear export receptor CRM1, for 3 h. The cells were fixed with 4% paraformaldehyde in PBS, permeabilized with 0.1% Triton X-100, and then blocked with 3% BSA. To detect STAT3 phosphorylation and phospho-STAT3 nuclear localization, cells were stained with phospho-STAT3 antibody (Cell Signaling Technology) for 2 h at room temperature. The cells were washed with PBST (0.1% Triton X-100 in PBS) containing 1% BSA and stained with Alexa Flour 488-conjugated secondary antibody (Thermo Fisher Scientific) for 1 h. Nuclei were stained with Hoechst 33258 (Thermo Fisher Scientific). The samples were observed by confocal laser scanning microscopy (CLSM, LSM 710, Carl Zeiss, Jena, Germany).
7
Bacterial Viability Assay using CLSM
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The untreated and HHP-treated E. coli and S. aureus cells were centrifuged at 8340 g for 10 min. The supernatant was removed and the pellet was washed twice with normal saline. Then, the cells were stained by a Live/Dead BacLight bacterial viability kit according to the manufacturer’s instructions (Molecular Probes Inc., Eugene, OR, United States). The Propidium Iodide (Cat. No. P8080) was purchased from Beijing Solarbio Science & Technology Co., Ltd. Bacteria solution was incubated with dye mixture (SYTO 9:PI = 1:1) at room temperature in darkness for 15 min, with a final concentration of 5 μM SYTO 9 and 30 μM PI. The cell suspension after staining was placed between a slide and an 18 mm square coverslip. Samples were examined under a Zeiss LSM710 CLSM (Carl Zeiss MicroImaging GmbH, Jena, Germany). In all cases, a 63× objective was used with immersion oil.
8
Propidium Iodide Staining of Skin Tissue
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For propidium iodide (PI) staining (Sigma-Aldrich) skin tissue specimens were fixed with paraformaldehyde and embedded in paraffin [72 (link)]. 5-µm sections were prepared. The slides were analyzed by confocal laser-scanning microscopy. Fluorescent staining was analyzed using a Zeiss LSM 710 CLSM (Zeiss, Jena, Germany) fitted with a Plan-Apochromat 63 × 1.4 objective as previously described [73 (link)]. The PI-stained slides were performed in triplicates.
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