Except for fluorescent labeled toxins (TcdA-PE/Cy5 and TcdA1 1874-Atto488) cells were subsequently fixed in 4% of formaldehyde solution, repeatedly washed and finally subjected to flow cytometry (FACScan flow cytometer; Becton Dickinson).
For nucleosomal DNA preparation, exponentially growing yeast cells were first cross-linked with formaldehyde, spheroplasted with zymolase and finally subjected to a MNase partial digestion to generate core nucleosomes containing DNA fragments of around 147 bp (see Additional File 1: Additional Methods).
In Step 4′, the hydrogen atom attached to N atom, for OONH system, is trapped by oxygen atom, which transferred to the carbon atom and produces methanol, whereas the chlorine atom, for OONCl system, combines with a hydrogen atom of H3C O moiety and finally forms formaldehyde and hydrochloric acid.
Cells were washed with PBS, then fixed with 4% formaldehyde in PBS for 20 min, and finally permeabilized with 0.2% Triton X-100 solution for 5 min.
Cells were washed with PBS and were then incubated with trypan blue (250 μg/mL) or PBS for 5 min. Cells were then washed again, trypsinated, centrifuged at 1600 rpm for 5 min, washed, and finally fixed with 2% formaldehyde.
Cells were next incubated with FITC or APC isotypes or FITC-CD4 or FITC-CD8 and APC-CD3 antibodies for membrane staining for 20 min at 4 °C in the dark and finally fixed with 1 % formaldehyde.
The bones were fixed in 10% normal buffered formaldehyde for 5 days, dehydrated by ascending concentrations of ethanol and finally embedded in methylmethacrylate (Technovit 7200, Heraeus-Kulzer GmbH, Wehrheim, Germany).
Acute (short-term) human exposure to formaldehyde causes discomfort; irritation of the eyes, nose, and throat; lachrymation; sneezing; coughing; nausea; and finally death.
Unreacted formaldehyde was quenched with 200 m M glycine, then cells were washed with PBS, and finally resuspended in SDS lysis buffer containing protease inhibitors.
After fixation in 4% formaldehyde and 70% ethanol, the VFs removed from the larynges were further processed, dehydrated, and finally embedded in paraffin wax and cut into 5 µm thick horizontal sections covering the whole thickness of each VF.
