Tcs sp8 confocal scanning system

Biofilm of C. albicans was developed by growing diluted overnight culture in YPD broth (OD_600nm=0.5) in a 24-well polystyrene plate for 24 hr in a static condition at 30 °C as described before (Bose et al., 2023 (link)). After, 24 hr of biofilm formation, the biofilms were either kept untreated or treated with 250 µM EDTA or mentioned concentrations of other metal chelators (DTPA, Aprotinin, TPEN, and CE), 8 µM MgSO₄, 8 µM ZnCl₂, 8 µM FeCl₂, 8 µM MnCl₂, 250 µM EDTA +8 µM MgSO₄, 250 µM EDTA +8 µM ZnCl₂, 250 µM EDTA +8 µM FeCl₂, and 250 µM EDTA +8 µM MnCl₂; and again, incubated for another 24 hr. After a total 48 hr of incubation, the supernatant was gently removed without disturbing the biofilm and the wells were washed with 1 X PBS twice. The biofilm so obtained was treated with 0.1% crystal violet and left for 20 min to stain at 25 °C. The plate was allowed to air dry and the bound dye was then re-suspended in 33% glacial acetic acid, and absorbance was recorded at 570 nm using a Perkin-Elmer plate reader. The experiments were repeated twice with biological triplicates. Similarly, biofilms were formed and treated in 8 well-chambered slides, images were obtained after staining with 1% acridine orange using Leica TCS SP8 confocal scanning system with where excitation at 483 nm and emission with 500–510 nm band-pass filter were used.

C. albicans cells were grown as described above in a 6-well cell culture plate with glass coverslips at 37°C for 24 hr. After 24 hr, the supernatant was discarded and washed with 1x PBS followed by staining with 1% acridine orange for 20 min in dark. Excess stain was removed by washing with 1x PBS, followed by fixation with 4% formaldehyde at 25°C for 30 min. Excess fixative was removed by washing thrice with 1X PBS. Finally, the glass coverslips were placed gently on the glass slide, and images were captured using Leica TCS SP8 confocal scanning system with an excitation wavelength of 483 nm and emission wavelength of 500 to 510 nm bandpass filter.

NPC grown on cover slide were fixed in 4% PFA, rinsed in PBS, permeabilized in PBT during 15 min, and saturated with PBS containing 10% of normal goat serum (PBG) for 1 h. Primary antibody targeting p21 was diluted in PBG, incubated overnight, and rinsed in PBS. Secondary antibodies diluted in PBG containing 1 μg/ml DAPI (4’,6-diamidino-2-phénylindole) were applied for 30 min at room temperature, washed in PBS, and coverslips were mounted in Fluoromount-G (SouthernBiotech). Imaging was performed using a Leica TCS SP8 confocal scanning system (Leica Microsystems) equipped with 405-nm Diode, 561-nm DPSS lasers. Eight-bit digital images were collected sequentially from a single optical plane using a Leica HC PL APO 20x, 40x or HC PL APO CS2 63x oil immersion lens (numerical aperture of 1.40) for cell analysis. Pictures with composite colors and the corresponding Tiff files were generated on ImageJ. DAPI and p21-positive nuclei count and area measurement was performed using the StarDist plugin on ImageJ/Fiji (Schmidt et al, 2018 ).
Whole E12.5 embryos and livers collected on 13.5 embryos were fixed in 4% paraformaldehyde (PFA, Sigma-Aldrich). Tissues were cryo-protected in 30% sucrose in phosphate-buffered saline (PBS), embedded in gelatin before freezing and kept at −80 °C until use. Serial 6- to 16-micron-thick sections were cut on a Leica CM 3050 cryostat.