Tcs sp5 confocal fluorescence microscope

Caco-2 cells were grown on glass on cover slips in a 24-well plate for 6 days post confluence in a normal tissue culture incubator. On day 6, media was changed and plates were placed at 1% O₂ or left under normoxia for 24 hr. Cells were fixed with ice-cold methanol for 15 min and washed with Tris buffered saline with 0.01% Tween 20 (TBS-T). Coverslips were then incubated in blocking solution of 3% BSA with 0.01% TBS-T for 30 min at room temperature followed by permeabilization using 0.3% Triton X-100 for 15 min at room temperature. After washing, coverslips were incubated with 0.01 mg/ml mouse anti-β1 integrin (Merck-Millipore, Germany) or the mouse IgG1 isotype control (Merck-Millipore, Germany) diluted in 3% BSA with 0.01% TBS-T at room temperature for 2 hr. Coverslips were washed with 0.01% TBS-T and incubated with secondary goat anti-mouse Alexa Fluor® 488-labeled antibody (Invitrogen, Germany) for 45 min at room temperature, protected from light. After washing, coverslips were embedded in ProlongGold + DAPI™ (Invitrogen, Germany). Microscopy was performed using a Leica TCS SP5 confocal fluorescence microscope with a HCX PL APO 40X 0.75–1.25 oil immersion objective. Gain settings were kept the same when acquiring images of cells grown under the two conditions.

Plasmodium
falciparum-infected RBC cultures were synchronized
at late stages by treatment in 70% Percoll (GE Healthcare, Uppsala,
Sweden) density centrifugation at 1070×g for
10 min. Then, 2.5 mg/mL of either DHP(G4)-MPA/hep or DHP(G4)-MPA-Rh/hep
was incubated with the synchronized pRBCs for 30 min at 37 °C.
After this time, samples were washed twice with phosphate-buffered
saline (PBS) and nuclei were counterstained with 2 μg/mL Hoechst
33342. For confocal microscopy analysis, samples were placed in a
μ-Slide eight-well chamber slide (ibidi GmbH, Gräfelfing,
Germany), and images were collected with a Leica TCS SP5 confocal
fluorescence microscope (Mannheim, Germany) using a 63× oil immersion
objective. Rhodamine was excited with a diode-pumped solid-state laser
at 561 nm and Hoechst 33342 with a diode laser at 405 nm, and the
fluorescence signal was collected in the range of 580–650 and
415–460 nm, respectively. To avoid crosstalk between the different
fluorescence signals, sequential scanning was performed.
Flow
cytometry targeting analysis was done in an LSRFortessa cytometer
(BD Biosciences, San Jose, CA, US) set up with the five lasers, 20-parameter
standard configuration. Hoechst 33342 and Rh signals were detected,
respectively, by excitation with 350 nm/60 mW and 561 nm/100 mW lasers
and emission collection with 450/50BP and 610/620BP nm bandpass filters.

To increase contrast during fluorescence imaging, Cy5 (free or RNA-labelled), Alexa Fluor 488 (Alexa 488; Thermo Fisher Scientific) or mCitrine (YFP), were included at final concentrations of 1 μM, 0.6 μM and 2 μM, respectively. Images were captured at room temperature using a Leica TCS-SP5 confocal fluorescence microscope and an HCX PL APO CS 63x (NA 1.40) oil immersion objective. Samples were illuminated with 488 nm (Alexa 488), 514 nm (YFP), or 633 nm (Cy5) lasers, with power and gain settings adjusted to give mean fluorescence intensity values of protein condensates of approximately 50–70% of the maximum 16-bit depth (∼32,000–46,000 a.u.). Images were typically captured with settings of 256 × 256 pixels at ∼ 98 × 98 × 98 nm (XYZ) resolution, 1400 Hz scan speed and a line average of 2.
Phase separation was initiated by mixing protein solutions with a buffer of lower ionic strength, as described above, and were left for 10 min to promote droplet growth. Phase separated solutions were then transferred to a 0.22 mm thick siliconized glass coverslip (Hampton Research), before sealing with 0.12 mm imaging spacers (Sigma) and a second siliconized glass coverslip. Samples were then left to equilibrate for approximately 50 min prior to imaging.

All tantalum specimens with adherent cells were rinsed with a D-PBS solution after the prescribed length of incubation and fixed with a solution of 4% methanol-free formaldehyde (Sigma-Aldrich, Oakville, ON, Canada) for 1 h in ambient conditions. The fixed cells were permeabilized in a 0.1% Triton-X 100 (Sigma-Aldrich) solution for 5 min. Specimens were rinsed with PBS and blocked with 2 mL of 1% (w/w) bovine serum albumin (BSA) (Sigma-Aldrich). F-actin microfilament staining was conducted by soaking specimens for 1 h in the deep red CytoPainter F-Actin stain (ab112127 Abcam, Cambridge, MA, USA) solution, which was diluted by a factor of 1000 in 1% BSA. A solution of 0.4 μg/mL of the 4′,6-diamidino-2-phenylindole (DAPI, Life Technologies, Waltham, MA, USA) was used to stain the DNA (5 min). All staining processes were performed in the dark to avoid photobleaching and the specimens were rinsed twice with 2 mL D-PBS after each stain application. The final solution contained four drops of Prolong Gold anti-fade reagent (Life Technologies). Specimens were kept refrigerated at 4 °C. A Leica TCS SP5 confocal fluorescence microscope (Wetzlar, Germany) at the University of Guelph, Ontario, Canada, was used to inspect stained samples with wavelengths in the range of 436 to 482 nm (for DAPI) and 650 to 700 nm (for CytoPainter F-Actin).

Cells were fixed with 4% paraformaldehyde for 15 min at room temperature (RT) and permeabilized with 0.1% Triton X-100 for 15 min at RT. After three washes with phosphate-buffered saline (PBS), the cells were incubated with 1% bovine serum albumin (BSA) for 1 h at 37 °C, followed by incubation with the primary antibody for 1 h at 37 °C, and then a fluorochrome-conjugated secondary antibody in the dark for 1 h at 37 °C. The cell nuclei were stained with 4′, 6-diamidino-2-phenylindole (DAPI) for 8 min. After three washes, the coverslips containing the stained cells were mounted onto the microscope slides using Fluoromount-G mounting medium (Beyotime, Shanghai, China). The fluorescence was visualized using a Nikon Ti microscope and a Leica TCS-SP5 confocal fluorescence microscope.