Sections were cut (5 um) and stained with hematoxylin and
eosin (H&E). Samples were then viewed using an Olympus BH-2-
RFCA (Japan) compound light microscope. Due to sectioning constrains, just the histological slides where the three sites of the gill
arch were visible and image analysis performed was considered for
further investigation. For analysis of lymphocyte density in the ILT,
two pictures at 1000 magnification were taken per section
examined (area ¼ 100 mm2). For consistency, the pictures were
taken in the same areas of the gill, one in the basal area and the
other in the outer area (tip) of the ILT. Lymphocyte numbers were
quantified manually using image analysis software (ImageJ e Kurt
De Vos, University of Sheffield, Academic Neurology http://imagej.
nih.gov/ij/plugins/cell-counter.html).