Free-floating sections (40 m) were washed in
0.1 M PBS, incubated in 0.1 M PBS containing 5% normal
donkey serum and 0.3% Triton X-100 for 1 h. They
were subsequently incubated overnight with Alexa
546-conjugated streptavidin antibody (1:500) in PBS
containing 2% normal donkey serum at 4◦C, washed
with PBS and then mounted sequentially in glass slides
using Vectashield. The mounted slices were evaluated
for fluorescence under settings for 546 nm emissions
on a confocal microscope (Olympus, USA).