Osmic acid

Kidney cortical tissues were cut into 1 mm³ pieces and fixed in 2.5% glutaraldehyde (pH 7.4, Spi-Chem, United States) for 2 h. After washed three times with 0.1 M phosphate buffer (pH 7.2) and fixed in 1% osmic acid (Ted Pella Inc., United States) at 4°C for 2 h, all the samples were gradient dehydrated in a graded series of ethanol. Subsequently, the samples were embedded in Epon-Araldite resin (Spi-Chem) for penetration and placed in a model for polymerization. After positioning, the ultrathin sections were collected for microstructure analysis. Counterstained using 3% uranyl acetate and 2.7% lead citrate. Finally, the samples were observed with a HT7800 transmission electron microscopy (TEM).

An agar solution of a 20 g/L was prepared with distilled water, heated to dissolve, and poured into a conical tube. After the EA cells were digested, they were added to a centrifuge tube and centrifuged at 1000 rpm/min for 5 min. The supernatant was discarded and resuspended in 5 mL of PBS. The suspended cells were added to an agar centrifuge tube and centrifuged at 2000 rpm/min for 15 min, and the supernatant was discarded. Then, 4% paraformaldehyde was added to fix for 15 min, the agar block was removed, and the cell mass was repaired with a knife, washed with PBS 3 times, fixed with 1% osmic acid (Ted Pella, Inc.) for 30 min, washed with distilled water 3 times, and dehydrated with 30%, 50%, 70%, 80%, 90%, 100% (I) and 100% (II) gradients for 2 min. The cell mass was soaked with resin (1:1 ethanol: resin for 60 min, 100% resin for 2 min) for 60 min and placed in a constant temperature oven (Liuyi, Beijing) at 60 ℃ for 2 h. The embedding agent was added in the capsule, and the label was placed. The agar block was moved to the center of the capsule, allowed to settle naturally to the bottom of the capsule, baked at 60 ℃ for 48 h, and sliced (Leica UC-7). The sections were stained with uranium acetate lead citrate and observed under an electron microscope (JEOL-TEM).

Germinated spore samples were fixed with 2.5% glutaraldehyde (Solarbio, Beijing, China) overnight at 4°C. Then, samples were fixed with 1% osmic acid (Ted Pella, Shanghai, China) for 1 hour and were dehydrated using a graded series of ethanol (30%, 40%, 50%, 60%, 70%, 80%, and 90%) for 10 minutes each treatment and two times 100% ethanol for 15 minutes. Following, the samples were dehydrated using tert-butyl alcohol: acetonitrile (2:1 and 1:1), followed by absolute acetonitrile for 10 minutes each treatment. Finally, the samples were observed with the scanning electron microscope (Phenom-World BV, Eindhoven, Netherlands).