Hcp 2 critical point dryer

Material of E. redactum was collected in a wild population in the campus of Shivaji University, Kolhapur, Maharashtra, India (voucher specimen: S.R. Yadav, 1 September 2019, MW). Samples of E. dalzellii and E. xeranthemum were collected near Abasaheb Marathe Arts and New Commerce, Science College, Ratnagiri, Maharashtra, India (voucher specimens: A. Chandore, 28 August 2019, MW). The material was fixed in 70% ethanol. For SEM, material was dissected in 70% ethanol and then transferred to 100% acetone using the following series: 96% ethanol (twice for 30 min), 96% ethanol: 100% acetone (1:1 v/v, 30 min), 100% acetone (three times 30 min). The material was critical point-dried using a Hitachi HCP-2 critical point dryer (Hitachi, Japan), then coated with gold and palladium using a Giko IB-3 ion-coater (Tokyo, Japan) and observed using a CamScan S-2 (Cambridge Instruments, London, UK) at the Laboratory of Electron Microscopy at the Biological Faculty of Moscow University.

EWs from half of the leaf were stripped by gum arabic; the other half was left untreated as control. Samples were air dried at room temperature. Before observation, small pieces of samples were fixed to aluminum sample holders, freeze dried (HCP-2 critical point dryer, Hitachi, Japan), sputtered with a thin layer of gold (IB5 ion coater, Eiko, Japan), and then observed under scanning electron microscope (SEM) (JEM-6380LV, JEOL, Japan).

Seedlings infected with A. tumefaciens were rinsed briefly with 0.9% saline. Seedling-associated A. tumefaciens cells were extracted by grinding the seedlings in 0.9% saline (1 ml for 10 seedlings from each well). The extracts or inoculation medium were serially diluted and spread on low-salt LB (0.5% NaCl) agar plates for incubation at 25°C for 2 days before counting colony-forming units (cfu). Agrobacterium cells on the seedling surface were observed by scanning electron microscopy (SEM, FEI Quanta 200). The samples were first mixed with fixation solution (2.5% glutaraldehyde, 4% paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.0) at room temperature overnight. The samples were rinsed with 0.1 M sodium phosphate buffer, pH 7.0 three times and post-fixed in 1% osmium tetroxide (OsO₄) for 4 h then rinsed with 0.1 M sodium phosphate buffer, pH 7.0 three times^{40 (link)}. Before viewing, samples were dehydrated in an ethanol series and dried with use of a Hitachi HCP-2 critical point dryer. SEM at 20KV was used for viewing.

Mitochondria morphology was visualized with transmission electron microscopy. Cells were fixed in 2.5% glutaraldehyde, post-fixed in 1% OsO₄, dehydrated in a graded series of ethanol solution, washed in acetone, and embedded in resin mixture. Sections were obtained by EM UC7 ultratome (Leica, Germany), and then stained by uranyl acetate and lead citrate. Images were taken by H-7650 transmission electron microscope (Hitachi, Japan). The ultrastructure of cilium in cells was observed using scanning electron microscopy. After dehydrated through an ethanol series and washed with pure ethanol, cell samples were inserted into HCP-2 critical point dryer (Hitachi, Japan) until dry, coated with gold-palladium in E−1010 ion sputter (Hitachi, Japan) for 4–5 min and observed in the SU-8010 scanning electron microscope (Hitachi, Japan).

For SEM, a single fragment ca. 5×5 mm was cut out from the leaf blade by a razor blade for each specimen. The fragment was taken from the central part of the leaf blade (equidistant from petiole/leaf base and leaf apex), equidistantly from midvein and leaf margin, between secondary veins. The dissected material was transferred from 70% ethanol to 100% acetone via 80% and 96% ethanol followed by an 1: 1 mixture of ethanol (96%) and acetone (100%). The material was critical-point dried using an HCP-2 critical point dryer (Hitachi, Japan). Dried samples were divided in two equal parts by a razor blade, which then were mounted onto stubs with different sides exposed, using double-sided sticky tape. The mounted specimens were coated with gold using an Eiko IB-3 ion-coater (Eiko Engineering, Japan) and observed using a CamScan 4DV (CamScan, UK) scanning electron microscope at Moscow State University.

For scanning electron microscopy (SEM), 5 seeds from 3 ears were collected from normal-size seeds (without any deformation and damage) and fixed in 2.5% glutaraldehyde in 0.1 M Sorenson buffer, pH 7.2. After having been washed with buffer, the samples dehydrated through ethanol series (30%, 50%, 70%, 96%, 2 × 100%). Then, CO₂ for critical-point-drying (Hitachi HCP-2 critical point dryer) was applied. Dry seed fragments were mounted on a SEM stub with carbon-conductive tabs and coated with gold and palladium using an Eiko IB-3 ion-coater (Eiko, Tokyo, Japan). Samples were observed and photographed under a JSM-6380LA SEM at 20 kV (JEOL, Tokyo, Japan).

For SEM, approximately 5 × 5 mm of VV tissue containing dark blue spots as a result of hematoxylin whole-mount staining were fixed using a fixing solution containing 2.5% glutaraldehyde and 4% paraformaldehyde in 0.1 M phosphate buffer. After six washes with 0.1 M phosphate buffer, the specimens were post-fixed with 1% osmium tetroxide (OsO₄) phosphate buffer (0.1 M) for 1 h at 4 °C. The tissue was then treated with tannic acid for 1.5 h at 4 °C. Subsequently, the specimens were dehydrated using an ascending ethanol gradient, immersed in 3-methylbutyl acetate, then dried using an HCP-2 critical point dryer (Hitachi, Tokyo, Japan). The dried specimens were sputter-coated using a Hitachi E-1030 ion sputter coater for 60 s, and then analyzed with the SEM (S-4100, Hitachi).