Tcs sp8 sr confocal microscope

The intracellular fluorescence imaging assay was performed as previously reported [18 (link)]. Briefly, parasites were seeded in 24-well plates at 5% parasitemia with 2% hematocrit. The cells were fixed and dropped onto cover slides coated with poly-L-lysine and perforated. The click reaction was then carried out. For confocal imaging of the intracellular probe, the coverslides were inverted on a slide and Leica TCS SP8 SR confocal microscope was used for rapid imaging. For immunofluorescence, the corresponding antibodies were incubated after the click reaction. A Dragonfly 200 Spinning Disk Confocal Microscopy was used for immunofluorescence imaging, and ImageJ software was used for semiquantitative analysis.

RAW264.7 cells were seeded into sterile glass-bottom dishes and stimulated by LPS for 3 h. Then cells were incubated with or without Cel-P at concentrations of 0.25, 0.5 or 1.0 μmol/L. Cells were fixed with 4% paraformaldehyde and permeabilized. Cells were treated with click reaction cocktail for 2 h, incubated with Hoechst dye, then washed twice with PBS.
After the click reaction, cells were further incubated at 4 °C overnight with antibodies against PKM2 (1:200) or HMGB1 (1:200), then with secondary antibodies and finally with Hoechst dye. Images were captured under a TCS SP8 SR confocal microscope (Leica, Munich, Germany).
In other experiments, RAW264.7 cells were seeded into sterile glass-bottom dishes, stimulated with LPS for 3 h, then incubated another 4 h with or without Cel (1 μmol/L). Cells were fixed with 4% paraformaldehyde, permeabilized, blocked with BSA, incubated at 4 °C overnight with antibody against HMGB1 (1:200), then incubated with secondary antibody (1:200), and finally with Hoechst dye (1:1000) and dye to label the cell membrane (1:1000). Cells were washed twice with PBS, and images were captured under the TCS SP8 SR microscope.

Pancreatic ductal cells were seeded on glass coverslips, fixed in 4% PFA for 15 min, and permeabilized with 1% triton X-100 for 10 min. They were then blocked in 4% BSA for 30 min at room temperature and incubated with primary antibodies (anti-CK19, Abcam, 1:200; PDX 1, Abcam, 1:100, anti-insulin, Abcam, 1:200; anti-glucagon, CST, 1:200) at 4°C overnight. Next, the cells were incubated with secondary antibodies conjugated with FITC or Cy5. The primary antibodies were replaced by PBS using a negative control. Positive cells were observed using a Leica TCS-SP8 SR confocal microscope.