Afs freeze substitution unit

Infected erythrocytes were prepared for transmission electron microscopy examination by high-pressure freezing—freeze substitution method as described65 (link). The cells were frozen in a high-pressure freezer (HPF010, Bal-Tec). Freeze substitution was then performed in a Leica AFS freeze substitution unit (Leica). The 100 nm-thick sections were examined. Knob widths were measured at their bases in the plane of the surrounding plasma membrane. At least 25 cells were investigated for each condition.

For high-pressure freezing suspension, cultured endothelial cells were harvested by filtering and immediately frozen in a high-pressure freezing apparatus (HPF010; Bal-Tec, Balzers, Liechtenstein). For subsequent freeze substitution, the material was kept at −85 °C for 60 h before slowly being warmed to 0 °C for a period of 18 h. Substitution was performed in an AFS freeze substitution unit (Leica, Bensheim, Germany). The sections were poststained with aqueous uranyl acetate/lead citrate, and images were captured with a Hitachi H7650 transmission electron microscope (Hitachi High-Technologies) operating at 80 kV.

5 DAG Arabidopsis seedling root tips were cut and high-pressure freezing/frozen by the high-pressure freezer (EM PACT2, Leica, Germany) prior to subsequent freeze substitution in acetone containing 0.4% uranyl acetate at −85 °C in an AFS freeze-substitution unit (Leica, Wetzlar, Germany). After gradient infiltration with increasing concentration of HM20, samples were embedded and ultraviolet polymerized for ultra-thin sectioning^{48 (link),78 (link)}. TEM analysis was performed by observing the ultra-thin section on the grids under the 80 kV Hitachi H-7650 transmission electron microscope (Hitachi High-Technologies Corporation, Japan) with a charge-coupled device (CCD) camera.

For scanning electron microscopy (SEM) observation, pollen grains were mounted on sample stubs. After dehydration in air for 30 min, pollen grains were coated with gold particles (EIKO IB-3). Pollen grains were then observed using a HITACHI S-3000N scanning electron microscope.
Immunogold labeling were performed essentially as described previously [44 (link)]. Anthers of qrt1^-/-, cog3^-/+qrt1^-/-, and cog8^-/+qrt1^-/- flowers were cut and immediately frozen in a high-pressure freezer (EM PACT2; Leica) followed by subsequent freeze substitution in dry acetone containing 0.1% uranyl acetate at –85°C in an AFS freeze substitution unit (Leica). Infiltration with HM20, embedding, and ultraviolet (UV)-polymerization were performed stepwise at –35°C. Immunogold labeling was performed with GFP (80 mg/mL) or EMP12 antibodies (40 mg/mL) and gold-coupled secondary antibody at a 1:50 dilution.
To visualize the structure of Golgi stacks by TEM, anthers of qrt1^-/-, cog3^-/+qrt1^-/-, and cog8^-/+qrt1^-/- flowers were cut and high-pressure frozen and substituted with 2% OsO4 in 100% acetone and infiltrated with Epon resin as described [57 (link)]. TEM examination was performed using a Hitachi H-7650 transmission electron microscope with a charge-coupled device camera (Hitachi High-Technologies) operating at 80 kV.

Following the same sample preparation process of SXT, the frozen samples were maintained under cryogenic conditions. After SXT processing, the samples were transferred into the Reichert–Jung AFS freeze-substitution unit (Leica, Vienna, Austria) and the temperature was increased from −140 °C to −90 °C. Pure acetone with 0.1% (w/v) uranyl acetate and 1% OsO₄ was added to the samples for 60 h at −90 °C. Samples were brought from −90 °C to −20 °C and finally to room temperature and then embedded in Spurrs’ Resin. Capsules were polymerized for 1 day at 60 °C. The polymerized capsules were cut by ultrathin sectioning and transferred to copper grids. The grids were stained with saturated uranyl acetate for 30 min and lead citrate for 5 min. The stained sections were visualized on a J FEG-TEM, FEI Tecnai G2 TF20 S-TWIN microscope (operating at 100 kV).