In addition we analyze by using conventional and lectin histochemistry methods the distribution and composition of glycoconjugates the in this epithelium in order to contribute to the understanding of the anatomy and functional morphology of the mollusks integument.2. Material and Methods2.1. AnimalsSeven individual adults of Haliotis tuberculata about 12cm in length were collected in different seasons along 2009 from different locations in the R��a of Vigo, (NW Spain). The habitat of Haliotis is infralitoral so a diver collected them from underwater rocks taking care not to damage the animals, and they were placed in seawater with aeration until they were processed into few hours.

After they were anesthetized by immersion in 5%MgCl2 in seawater [30], small pieces of the foot were cut from the medium edge part of the animal body taken at the same time the lateral and ventral part of the foot as it is showed in our previous paper [29]. We analyzed in the same section the lateral part of the foot integument named side foot and the sole foot which is the surface of locomotion in contact with the substrate. All procedures for animal experimentation were approved by the animal care and use committee of the Regional Government of Galicia (Xunta de Galicia) and conformed to the guidelines of the European Community.2.2. Light MicroscopySamples were fixed in formol Baker (Prolabo) for 24�C48h at room temperature, washed in tap water, and embedded in paraffin. Sections (8��m thick) were deparaffinized in xylene, rehydrated with graded ethanol, and subjected to the following histochemical procedures for the identification of glycoconjugates.

2.2.1. Conventional Histochemical Techniques Sections were stained with alcian blue (AB, pH 1 or 2.5, Sigma) to demonstrate acidic glycoconjugates, and high iron diamine (HID) combined with alcian blue (HID/AB) for separating sulphated and carboxylated glycoconjugates. As a control some sections were desulphated before stained with AB or HID. Desulphation is a sequential process of methylation and saponification specifically applied to remove sulphate-ester groups [31].Samples were also subjected to chemical method using periodic acid-Schiff reactive (PAS, Merck) which is positive for glycoconjugates containing neutral sugars and/or sialic acid. In histochemistry, the presence of neutral monosaccharide residues means that they do not have sulphate-ester, carboxylic acid, or nitrogen-containing functional groups. In order to know if the PAS-positive Cilengitide compounds were glycogen, an amylase test was carried out by using rat liver sections as a positive control. All staining protocols were performed according to Kiernan [31] and Molist et al. [32].2.2.2.