Cells were then washed with 1 × PBS, lysed with reporter lysis buffer (Promega E397a) and left at −20 oC overnight.
The cell extract was diluted in the ratio of 1 : 1 with sample buffer (TEFCO, Tokyo, Japan) and left at room temperature for 10 min.
After centrifugation, packed cells were suspended in a Red Blood Cell Lysing Buffer (Sigma-Aldrich) and left at room temperature for five minutes to lyse red blood cells.
Animals were then transferred to a solution containing 2.5% glutaraldehyde, 2% paraformaldehyde, 1% sucrose, 1 mM CaCl2 in 0.05 M NaCacodylate buffer pH 7.36 and left at 4°C until ready for processing.
Dried samples were suspended in lysis buffer containing protease inhibitors and left at 4°C for 30 minutes.
Red blood cell lysis buffer (RBCly) was added to the hepatocyte suspension and left at room temperature for 3 min before inactivation with PBS.
Fresh MES KCl buffer was then added to the pellets, which were resuspended and left at room temperature for 20 min.
The pellet was resuspended in infiltration buffer (10 mM MES, 10 mM MgCl2, 150 μg acetosyringone/ml and 2% sucrose) and left at room temperature for three hours.
Briefly, 210 μl of sample containing transport medium spiked with 6 μl of IPC RNA were mixed with the provided lysis buffer and left for at least 10 min at room temperature.
Thereafter, 500 mg of the dried grids was immersed in 50 mL of distilled water or rumen buffer and left standing at 20°C for 24 h.
