Blood collection was performed at day 38 following two
booster doses administered at day 14 and day 28. After rinsing the
stained grids with 0.1% BSA in PBS for 2 min, the grids were
preincubated in Solution 2 (0.01% fish gelatin in PBS), transferred
to a drop of gold-conjugated goat anti-mouse antibodies (diluted
1:40 in Solution 2) for 60 min, washed six times with Solution 2
and twice with PBS for 10 min each. The grids were fixed in 2%
glutaraldehyde for 15 min, then washed with PBS four times for
10 min and stained with 1% ammonium molybdate for 10 sec.
Two negative controls were concurrently tested using antibodies
derived from a non-immunized mouse or rotavirus instead of
PMMoV.