Confocal laser scanning microscope

HFFs were cultured on 12 mm coverslips in complete medium 24 hours before the experiment. Tachyzoites were added to HFFs (MOI = 2) and invasion in the absence (0 mM) or presence (2 mM) of glutamine was allowed for 6 hours at 37°C in the incubator with 5% CO₂ and then washed by PBS three times and stained with anti-SAG1 (1∶10,000) for 15 minutes, which labels extracellular parasites before 4% paraformaldehyde fixation. Coverslips were then blocked for 30 minutes and incubated in Alexa Fluor 555 (1∶1000) for 2 hours. A Zeiss confocal laser scanning microscope (LSM) with a 40× objective was used to count the number of parasites and total of 15 fields per coverslip were examined. The ratio of intracellular tachyzoites to total tachyzoites in the examined field on the coverslip is defined as infection efficiency.

To quantify the expression of GmPHR1 in overexpression lines, shoots of GmPHR1-OE transgenic plants and wild type plants were ground in liquid nitrogen, and total protein was extracted in Pierce IP buffer (Thermo Scientific, Waltham, MA, USA) amended with 1 mM PMSF, 5 mM MG132, and 1× protease inhibitor cocktail (Roche, Basel, Switzerland). Total protein (20 µg) was loaded onto 12% SDS-PAGE gels for electrophoresis and then transferred to NC membranes (Bio-Rad, Hercules, CA, USA) using Trans-Blot Turbo (Bio-Rad). The blotting procedures were conducted according to Zhong et al., 2018 [13 (link)]. After washing three times, bound antibodies were visualized with ECL substrate (Millipore, Burlington, MA, USA) using the ChemDoc XRS system (Bio-Rad). The dilution ratio for mouse anti-flag (Sigma-Aldrich, St. Louis, MO, USA) was 1:3000.
To quantify deformed root hairs, transgenic GmPHR1-OE soybean seeds were germinated along with wild type soybean seeds in sterile vermiculite, inoculated with 50 mL RFP labeled Bradyrhizobium BXYD3, and supplied with nitrogen free nutrient solution. Root hair phenotypes were recorded using a confocal laser scanning microscope (LSM; Carl Zeiss, Oberkochen, Germany) five days after rhizobium inoculation [36 (link)]. The ratio of deformed root hairs was calculated as the number of deformed root hairs to the total number of root hairs.

Before the staining step of the confocal laser scanning microscopy (CLSM) assay, C. albicans biofilm formation was performed in glass-bottom cell culture dishes (NEST Biotechnology Co., Ltd., Wuxi, China) using the method described above. The biofilms incubated without magnolol were regarded as the live controls, and those incubated with isopropyl alcohol for 60 min were regarded as the dead controls. The magnolol solution was diluted to achieve 80, 160, and 320 μg/mL.
Rapid immunofluorescence staining was performed using the LIVE/DEAD FungaLight Yeast Viability Kit (Molecular Probes, Inc., Eugene, OR, USA). Live yeasts with intact cell membranes were stained fluorescent green by SYTO9, whereas dead yeasts with damaged membranes were stained fluorescent red, indicating the penetration of propidium iodide. According to the kit manufacturer’s protocol, 500 μL of stain solution was added to each biofilm dish and incubated in the dark at room temperature for 30 min. The biofilms were observed under a confocal laser scanning microscope (Carl Zeiss, Inc., Oberkochen, Germany). Then, three-dimensional (3-D) reconstruction was carried out using Leica Application Suite X (LAS X) software. At least three random visual fields were chosen in each dish, and three duplicate culture dishes for each group were observed.

A549 cells were subcultured in a 6-well plate and incubated at 37 °C 5% CO₂. After sample preparation by fixation, permeabilization, and blocking, the slides were incubated with primary antibody diluted in 3% BSA at 4 °C overnight. Following primary antibody incubation, the slides were then washed three times and incubated with conjugated secondary antibodies in 3% BSA for 1 h at RT. The slides were washed three times with PBST and counter stained with DAPI (Vector Laboratorise, Burlingame, CA). Immunostained slides were imaged using a confocal laser scanning microscope (Carl Zeiss, Thornwood, New York) and analyzed with Zen software.

BM-MSCs were washed with PBS and fixed with 4% paraformaldehyde for 40-60 minutes. After permeabilization and block, BM-MSCs were incubated with fluorescein isothiocyanate-conjugated phalloidin (Thermo Fisher, Waltham, USA). The cell nuclei were stained with DAPI (Sangon Biotech, Shanghai). The stained cells were visualized with a Zeiss Confocal Laser Scanning Microscope (Oberkochen, Germany).

Immunofluorescence was conducted as previously described [10 (link)]. The primary antibodies were used as follows: NF-κB (cell signaling), MMP-9 (Abcam), and HO-1 (Abcam). The representative images were obtained using a confocal laser scanning microscope (ZEISS, Dresden, Germany).