2.2. Detection of carbohydrate moieties in gill filaments and hemocytes
of B. azoricus
The combined Alcian blue and Periodic-Acid Schiff (PAS) staining
method (Woods and Ellis, 1994–96) was utilized to determine the
distribution of glycoproteins in paraffin tissue sections of gills and in
hemocytes of B. azoricus. In brief, after deparrafinization and serial
rehydration, tissues sectionswere transferred to distilledwater and then
stainedwithAlcianblue for 20min. After rinsingwithdistilledwater, the
sections were treated with periodic-acid for 10 min and subsequently
stainedwith Schiff's reagent for 20min. The sectionswere finally rinsed
thoroughly with distilled water, dehydrated and mounted for light microscopy. After the Alcian-PAS staining and in order to enhance the
color of acidic (blue) and neutral (magenta) carbohydrate moieties, the
nuclei staining with haematoxylin was minimal (1–2 min) in those
tissue sections not meant to be visualized under fluorescent light. For
the detection of glycoproteins in hemocytes, mussel hemolymph was
collected directly onto a positively charged microscope slide (Super-
Frost® Plus). Hemocytes attached immediately to the glass slide
and were treated as for gill tissues, after a short fixation with
10% buffered formalin.