Thiocarbohydrazide

Tissues designated for electron microscopy were perfusion fixed for 10 minutes (2% paraformaldehyde and PBS at pH 7.43). The pleura was stained with ruthenium red, lung tissue was submerged in 1% ruthenium red Sigma (Steinheim, Germany) and incubated for 24 hours at 4°C and post-fixed with 1% GA [50 (link)]. The post-fixation and ruthenium red labeled samples of the pleura were subjected to reduced OsO4-thiocarbohydrazide-OsO4 (rOTO) method in conjunction with lead aspartate was used for block staining. Briefly, tissue blocks were stained with 1.5% potassium ferrocyanide (Sigma P-8131) and 2% osmium tetroxide (OsO4) in distilled water for 1 h. After washing in distilled water, the samples were incubated in 1% freshly filtered thiocarbohydrazide (Sigma 88535), followed by three rinses with distilled water. Subsequently, the samples were incubated in 2% OsO4 in distilled water for 30 min at 4°C and then washed with water overnight at 4°C. Finally, the sample were washed in 0.1 M cacodylic buffer pH 5.5 for 20 min., incubated with lead aspartate (Merck 11195,) for 30 min at 60°C. The samples were then dehydrated in a in a series of ethanol dilutions (70%, 95% and 100%). Samples were embedded in Epon (Serva, Heidelberg, Germany); 70 nm ultrathin sections were analyzed using a Zeiss EM10 digital transmission electron microscope (Zeiss, Oberkochen, Germany).

Following overnight fixation in 2.5% EM grade glutaraldehyde at 4 °C, samples were washed (3 × 5 min) in 0.1 M sodium cacodylate buffer prior to OTO (osmium tetraoxide–thiocarbohydrazide–osmium) processing. Briefly, this method included post fixation in equal volumes of 4% osmium tetraoxide (Agar Scientific Ltd. Essex, UK) and 3% potassium ferrocyanide (Sigma-Aldrich, St. Louis, MO, USA) for 60 min on ice. Following post fixation, samples were rinsed (3 × 5 min) in dH₂O before incubating with thiocarbohydrazide (Sigma-Aldrich, St. Louis, MO, USA) for 20 min. Additional dH₂O washing steps (3 × 5 min) were applied before incubation in 2% aqueous osmium for 30 min at room temperature. Following OTO processing, bacterial samples were stained in 1% aqueous uranyl acetate (1 h at 4 °C) followed by lead aspartate (200 µM) for 30 min in the dark. Between these steps, washing with dH₂O was performed. After the final washing step, bacterial samples were dehydrated and dried as previously outlined.

Paraffin embedded mouse lung blocks used for confocal microscopy were dewaxed, rehydrated, and processed for scanning electron microscopy. Dewaxed mouse lung blocks were washed with HemoDe^® (EMS) to remove excess paraffin, followed by rehydration through a graded series of alcohol and distilled water mixtures⁵³ . To enhance specimen contrast and surface conductivity, rehydrated mouse lung blocks were first incubated with 1% osmium tetraoxide (OsO₄; EMS) and 1.5% potassium ferrocyanide (EMS) in 0.1 M SCB, pH 7.2, at room temperature for 2 hours, rinsed with 0.1 M SCB, incubated with freshly prepared 1% thiocarbohydrazide (Sigma) for 30 minutes, and fresh 1% OsO₄ in 0.1 M SCB for an additional hour. After osmium impregnation, en bloc staining with 4% uranyl acetate (EMS) was conducted at 4 °C overnight, followed by freshly prepared Walton’s lead aspartate staining en bloc at 60 °C for 1 hour^{54 (link),55 (link)}. Lung blocks were dehydrated through a series of graded alcohol and processed for critical drying using a Leica EM critical point dryer (Leica Microsystems Inc.). Mouse lung samples were mounted on 15 mm specimen stubs (EMS) and viewed with a Hitachi SU-8010 field emission scanning electron microscope (Hitachi High Technologies USA) at 5 kV without further coating.

Wolffia plantlets were fixed with 2% (w/v) glutaraldehyde (Sigma-Aldrich, G7651) and 2% (w/v) paraformaldehyde (Sigma-Aldrich, P6148), 0.1 m phosphate buffer (81 mm Na₂HPO₄ (Sigma-Aldrich 71649), and 19 mm NaH₂PO₄ (Sigma-Aldrich 71507), pH 7.4, for 2 h at room temperature, and then held overnight at 4°C. The next day, the samples were washed with 0.1 m phosphate buffer for three times and postfixed in 2% (w/v) OsO₄ (Ted Pella, 18459) and 1.5% (w/v) potassium ferrocyanide (Sigma-Aldrich, P3289) for 2 h at 4°C. Following five washes with phosphate buffer and distilled H₂O, samples were put into 1% thiocarbohydrazide (Sigma-Aldrich, 223220) and followed by another postfixation in 1% (w/v) OsO₄, and three times of washing with distilled H₂O were also included after each step. At last, samples were dehydrated through a graded alcohol series and embedded in Spurr's resin (SPI Supplies, 02680-AB).
Resin blocks were used for micro-CT, and the pictures were taken by Xradia Context (Zeiss) or SkyScan 1272 (Bruker). Next, selected resin blocks were cut into 70-nm sections using an ultramicrotome (Leica Microsystem, UC7) referring to the micro-CT data for transmission electron microscopy (TEM) (JEOL, JEM-1400).