Spi pon 812 resin

All samples were prefixed in 2.5% glutaraldehyde (v/v in 0.1 M phosphate buffer, pH 7.2) for 2 h, and then rinsed 3 times with 0.1 M phosphate buffer (pH 7.2). They were post-fixed in 1% OsO₄ for 2 h, followed by three 15 min rinses with phosphate buffer. Afterwards, the samples were dehydrated through an acetone series (30, 50, 70, 90, 100, 100, and 100%) (v/v in dd H₂O) at room temperature, samples were incubated for 20 min at each concentration. Then the samples were infiltrated in a graded scale of 3:1, 1:1, 1:3 (v/v) acetone/SPI-PON 812 resin and, as the last step, in 100% (v/v) SPI-PON 812 resin (SPI Supplies, West Chester), for 12 h per step. Samples were embedded in SPI-PON 812 resin and polymerized at 60 °C for 48 h. Ultrathin sections (80 nm) were prepared using an EM UC7 Ultracut ultramicrotome (Leica, UC7). Sections were observed and photographed using a transmission electron microscope (Hitachi H-7650) at an accelerating voltage of 80.0 kV. For ER-mitochondria quantification, the shortest distance between mitochondria and associated ER membrane was measured and at least 20 mitochondria from three independent biological samples were analysed.

For chemical fixation of Arabidopsis roots, 5-mm root tips of 5-day-old Arabidopsis expressing UBQ10::GFP:Exo84c and VAP27-3p::VAP27-3:mCh following 1 μM Conc A treatment were collected and checked with confocal microscopy, then they were prefixed as in 2.5% glutaraldehyde (v/v in 0.1 M phosphate buffer, pH 7.2) for 2 h. Then the samples were rinsed with 0.1 M phosphate buffer for three times, 15 min each. The samples were post-fixed in 1% OsO₄ for another 2 h, and rinsed with 0.1 M phosphate buffer for three times as before. All the samples were dehydrated using an acetone gradient series of 30, 50, 70 and 90%, with a 20 min exposure to each acetone gradient and ending with the dehydration step in 100% acetone for three times (15 min each). The samples were then infiltrated in a graded scale of 3:1, 1:1, 1:3 (v/v) acetone/SPI-PON 812 resin and 100% SPI-PON 812 resin (SPI Supplies, West Chester, PA, USA) at the last step, each step was performed for 12 h. Samples were finally embedded in SPI-PON 812 resin and polymerized at 60 °C for 48 h. For transmission electron microscopy (TEM) analysis, the embedded samples were sectioned on an EM UC7 ultramicrotome (Leica, Germany). Ultrathin sections (80 nm thick) were stained with 2 % uranyl acetate and lead citrate and viewed using a TEM (Hitachi H-7650, Japan) at 80 kV.