Airyscan confocal laser scanning microscope

CRC cells were seeded on chamber slides until adhered and cocultured with F. nucleatum (MOI of 100:1) for 2 h. After washing with PBS, the cells were fixed with 4% paraformaldehyde for 15 min at room temperature (RT). The slides were incubated with 0.2% Triton X-100 for 15 min and blocked in 3% BSA for 30 min. After washing by PBST three times, the cells were incubated with primary antibody against YAP (Proteintech, China) at RT for 2 h. Then the slides were washed by PBST three times and incubated with secondary antibodies and DAPI at RT for 1 h. The cells were fixed with Gold Antifade Reagent (Invitrogen, USA) and imaged using LSM 800 with Airyscan confocal laser-scanning microscope (Zeiss, Germany).

Living B. subtilis was stained with the LIVE/DEAD BacLight Bacterial Viability Kits (Invitrogen) based on the instructions provided by the reagents’ manufacturers. To test the capability of PCH₂-LA-UCNPs for manipulating the lysozyme activity in living B. subtilis system, PCH₂-LA-UCNPs were mixed with active lysozyme for 10 min. The mixture was incubated with living B. subtilis. The resultant B. subtilis samples were irradiated with 980 nm NIR laser (3 W cm⁻², 4 min break after 2 min irradiation) for a certain time (0 min and 10 min) and subsequently imaged with the LSM 880 with Airyscan confocal laser scanning microscope (Carl Zeiss GmbH, Jena, Germany). The adjusted settings of the instrument were listed as below: PI channel Wavelength (543 nm), laser power (2.0%), pinhole (90.1), gain (600); SYTO 9 channel wavelength (488 nm), laser power (5.0%), pinhole (90.1), gain (600), T PMT gain (380). The intensity minimum to black and intensity maximum to white value of images were 32 and 225, respectively.

Embryos were dissected at E11.5 and fixed in 4% paraformaldehyde solution (PFA) (pH 7.4) at 4°C overnight. After the tissues were soaked in PBS/1% Triton X-100 for 20 min and rinsed with PBS several times, they were blocked with PBS (0.3% Triton X-100, 5% normal donkey serum and 1x PBS) overnight at 4°C. The tissues were incubated overnight at 4°C with primary antibodies against phospho-ERK (Cell Signaling Technology, 4,370; 1:100), phospho-AKT (Cell Signaling Technology, 8,272; 1:100), and phospho-PLCγ (Cell Signaling Technology, 8,713; 1:100). The tissues were washed 3 times with PBS for 30 min each, incubated with Cy5-conjugated donkey anti-rabbit secondary antibody (Jackson, 1:500) overnight at 4°C and washed several times with PBS. Images of stained tissue were captured using a Zeiss-LSM880 with Airyscan confocal laser scanning microscope and ZEN ImageJ software (Zeiss, Germany). The details about how the measurements were conducted on the confocal images are described in the literature (25 (link)). ImageJ was used to quantify the amount of fluorescence as mean gray value of pERK, pAKT, PLCγ and compare the results among four groups. ImageJ was also used to determine the number of stained cells by phosphorylation of histone H3 (PHH3) to compare the results among these groups.