Hearts were rapidly removed, transferred into 10 ml 0.9% NaCl solution containing 1,000 U heparin, and connected to a cannula in a Langendorff perfusion system.
The supernatant was removed, transferred to a new centrifuge tube and re-centrifuged at 16,000 g for 10 min at 4°C to facilitate removal of the lipid layer overlying the supernatant.
The pieces of each sample were removed, transferred to 2.5% glutaraldehyde in sodium cacodylate buffer 0.1 M for transmission electronic microscopy analyses or to Zamboni fixative solution [41] for light microscopy.
The upper aqueous phase was then removed, transferred to a fresh tube, and 500 μL of isopropyl alcohol was added.
At each time point, the buffer was completely removed, transferred into the well of a microtiter plate and replaced by fresh buffer solution.
The intact chloroplast layer, identified by microscopy, was removed, transferred to SDS-PAGE cocktail, boiled for 10 min and stored at -20°C.
When the tumour size reached 100 mm, it was aseptically removed, transferred into ice-cold DMEM, and cut into 1 mm-sized fragments.
Trigeminal ganglia were removed, transferred into HBSS and enzyme-digested by incubation with papain (Worthington), collagenase type II (CLS2) (Worthington), and dispase type II (MB).
The aqueous phase was removed, transferred to a separate tube containing 500 μL of isopropanol (Sigma), gently mixed and centrifuged for 10 mins at 12,000 × g at 4 °C.
The microcentrifuge tube containing the benzethonium hydroxide and trapped CO2 was then removed, transferred to a scintillation vial, and the radioactivity counted.
