Microstructure of the fruit was measured according to the re-
ported method (Tironi et al., 2007), with some modifications.
At 20
C, frozen litchi samples (pericarp/pulp) collected from each
storage time (1 day, 60 days, 180 days) were cut into small pieces
(approximately 4 4 8 mm). These samples were then placed on
the specimen plates in the chamber of a microtome (Leica CM-
1900, Germany, equipped with a freezing system) at 25
C, then
were coated with Tissue Freezing Medium (TFM, Leica Microscopic
Systems Inc., Germany) layer by layer, followed by cryofixation for
2 h. The fresh litchi samples (pericarp/pulp) were cut into small
pieces (approximately 4 4 8 mm) in a room at 4
C, and put into
ethanol solution (13e17 mol/L) for gradient dehydration (1 h/
gradient). After that they were sealed in a glass bottle filled with
glycerol solution (10 mol/L) for 3 h. Bubbles in the bottles were
periodically removed using a microsyringe, and these samples were
placed overnight at 20
C. On the second day the samples were
coated with TFM layer by layer in the chamber of the microtome,
until the cryofixation process (2 h) was complete.
After cryofixation, 3e5 tissue sections (20
m
m) were obtained
from IF, AF and fresh litchi samples using the microtome. These
tissue sections were placed on glass slides fixed in methanol for
30 s, and then washed gently with distilled water, followed by
staining with toluidine blue (0.1 g/L) for 5e10 s, washed again and
air dried. Finally the microstructure of litchi tissue sections (peri-
carp/pulp) was observed in a microscope equipped with a CCD RGB
camera (MACC-C71, Sony, Japan).