Propylene oxide

Cerata, rhinophores, and tentacles from 0-day fasted, 15-day fasted (unbleached), and 50-day fasted (bleached) individuals were excised and fixed in an aldehyde fixing solution (2% PFA, 2% glutaraldehyde, 50 mM HEPES, 33 g/L Instant Ocean). Subsequently, the samples were washed with distilled water and post-fixed with 1% osmium tetroxide (Nisshin EM) at 4°C for 1 hour. Following further washing and dehydration using an ascending ethanol series, the tissues were replaced with propylene oxide (Wako) and ultimately embedded in epoxy resin (Quetol 812, Nisshin EM). Resin blocks were sectioned using an ultramicrotome (Leica, Ultracut UCT). Semi-ultrathin sections (500-nm thickness) were stained with toluidine blue (Diagonal GmbH & Co. KG.) and observed under an optical microscope (KEYENCE BZX-810). Ultrathin sections (60-nm thickness) were stained with 4% uranyl acetate solution (Bio-Rad) and Reynolds' lead citrate (Wako), followed by examination using a transmission electron microscope (JEOL JEM-1010). Transmission electron microscopy (TEM) images were overlapped and merged using Image Composite Editor Version 2.0.3.0 (Microsoft). Scale bars were manually generated referencing the original images.

HeLa 229 cells were cultivated on sterilized glass slides and the autophagy was induced under nutrient-starvation condition with or without 10 µg/ml rTSST-1 in the presence of lysosomal protease inhibitors. At 4 h after induction, the cells were fixed with 4% paraformaldehyde, 1% glutaraldehyde (Wako) in PBS, and then post-fixed with 1% osmium tetroxide (Heraeus Chemicals, Port Elisabeth, South Africa) in 0.1 M phosphate buffer (pH 7.4). The samples were dehydrated through a graded series of ethanol (Wako) and propylene oxide (Wako) at room temperature, and embedded in Epon 812 resin (TAAB Laboratories Equipment Ltd., Berkshire, UK). They were then polymerized with the resin in gelatin capsules (No. 0; Eli Lilly Co., Indianapolis, IN) at 60°C for 48 h. After polymerization, the samples on glass slides were transferred to resin block. Ultra-thin sections (70–80 nm) were cut with a diamond knife, stained with Sato’s lead citrate [26] (link) and uranyl acetate (Merck, Darmstadt, Germany), and observed under a transmission electron microscope JEM 1250 (JEOL Ltd., Tokyo, Japan) at 80 kV.

Cellular samples were fixed in 0.9% NaCl, 1% glutaraldehyde (Wako) for 1 h at 4 °C. Cells were pelleted and re-suspended in 1 mL of a 37 °C pre-warmed 1.1% agarose solution (Bio-Rad). After centrifugation (3000 rpm at 37 °C), pellets were placed at 4 °C and cut into small specimens, washed in 5% sucrose PBS (Wako) and post-fixed with 5% sucrose, 1.5% osmic acid (Nisshin EM) in 100 mM phosphate buffer (Wako) pH 7.3, for 1 h at 4 °C. Post-fixed specimens were washed in 5% sucrose PBS and dehydrated in a graded series of ethanol (Wako) and propylene oxide (Wako), then embedded in epoxy resin EPON 812 (TAAB Laboratories). Ultrathin sections were obtained with an ultra-microtome (Leica EM UC7) and subjected to double staining with a uranyl-acetate (Wako) and lead-citrate (Nacalai Tesque). Samples were observed under a JEM-1400Plus Electronic Microscope (JEOL). TEM were analyzed by collecting images of at least 3 representative areas (RA) at magnification of ×500. One RA contained 10–20 nucleated cells. Then representative images were taken at higher magnifications.