Various histochemical techniques have been employed to distinguish the different cell layers within the epidermal generations of Natrix piscator.
The oberhautchen and the thin superficial areas of the beta-layer contain both alpha- and beta-keratins and give moderate reactions with Papanicolaou's stain for keratin, for protein bound NH2 groups, basic proteins, and calcium; strong reaction for cysteine bound sulphydryl (SH) groups and very strong reaction for disulphide (SS) bonds of cystine. The remaining bulk of the beta-layer gives weak reactions with Papanicolaou's stain for keratin, for NH2 groups, basic proteins, SH groups and SS bonds. The mesos and the alpha-layers stain strongly with Papanicolaou's stain for keratin, for NH2 groups, basic proteins, sulphydryl groups and calcium, and weakly for mucopolysaccharides. The mesos layer also gives strong reactions for disulphide bonds and might contain both alpha- and beta-keratins.
The cells of the lacunar tissue are metabolically active as indicated by their normal nuclei and positive reaction for RNA. These cells contain granular glycogen, small amounts of acid mucopolysaccharides, NH2 and sulphydryl groups. The underlying layer of clear cells remain weakly or almost unstained with various histochemical techniques.
The inner generation could roughly be divided into two layers–the presumptive beta-layer and the presumptive alpha-layer. High RNA contents in the presumptive beta cells indicate the active polymerization of keratin in this layer. These cells give weak reactions for protein bound NH, groups, basic proteins, SH groups and calcium. The remaining cells of the inner generation are not easily distinguished and contain small amounts of RNA.
The differentiation of outer and inner generation in the hinge region is not clear. The thin outer oberhautchen and the beta-layer give very weak reactions for NH, groups, SS bonds, SH groups and calcium and stain orange-red with Papanicolaou's stain, bluish-black with Verhoeff's elastin stain and pinkish red with Mallory's triple stain. The alpha-layer resembles in its staining properties the alpha-layer of the scale region.
A large number of eosinophilic granular cells could be observed in the lower layers of the epidermis, especially in the hinge region and in the dermis.