The study was conducted on adult lizards of the green anolis (A. carolinensis, n=4) maintained in a terrarium at 25-33℃ as previously indicated [5]. The tail was amputated by twisting it to exploit the natural fracture plains present in the tail (autotomous planes) [1]. Despite the little bleeding, some blood was aspirated from the amputation surface of the stump with a Pasteur pipette and fixed into an eppendhorf test tube. Also, pieces of the detached normal tail were sampled for comparison with regenerated tissues (see later).

The animals were left to regenerate their tail at room temperature (25-30℃) in cages. In the first sampling, stumps of healing tails were collected after 4 days post-amputation (wound healing stage), and the same animals were re-sampled after 4 weeks when regenerating tails of 3-5 mm in length were formed. The tissues were immediately fixed for the following immunocytochemical study. Also the blood derived from the second amputation was collected in the fixative solution as indicated above.

Tissues were fixed at 0-4℃ in fresh 4% paraformaldehyde in 0.1 M phosphate buffer at pH 7.4 for 7-8 hours, rinsed in buffer, dehydrated in alcohol, and embedded in the resins Lowicryl or LR-White. In three cases also the blood from normal and regenerating lizards (3-4 days after tail amputation) was fixed for 1 hour and then centrifuged in an eppendhorf test tube for 3 minutes at 10,000 g. The supernatant was discharged and the pellet was dehydrated and embedded as indicated above.

The embedded tissues or blood were sectioned using an ultramicrotome, and semithin sections (2-3 µm thick) and thin sections (70-90 nm thick) were collected, stained in 1% toluidine blue for the histological examination or were utilized for the immunohistochemical study. The antibody against BD-27 was raised in a rabbit immunized with a 35 amino acid long peptide (DLECQKAKGLCLHQRCRRPWRSVGTCNNLQHHCCQ) for A. carolinensis beta-defensin-27 (Fig. 1) [6]. The peptides were conjugated to Key Limpet Haemocyanin before injection into rabbits, and the derived antibodies present in the immune serum were purified by affinity chromatography (Davids Biotechnologie, Regensburg, Germany). The reactivity of the immune serum to the antigenic peptides was tested by the manufacturer using the enzyme-linked immunosorbent assay-test.

Light microscope immunocytochemistry was performed incubating the sections overnight at 0-4℃ with the beta-defensin-27 or cathelicidins antibodies diluted 1:200 in buffer (Tris 0.05 M at pH 7.6 containing 1% bovine serum albumin). In control sections, the primary antibody was omitted or pre-absorbed for 24 hours at 4℃ mixing 3:1 of the solution containing the peptide antigen with the purified antibody solution. After rinsing in buffer, the sections were incubated for 60 minutes at room temperature in a fluorescein-conjugated anti-rabbit antibody (1:100, Sigma, St. Louis, MO, USA), rinsed in buffer, mounted in 10% glycerol in phosphate buffered saline, and observed under a fluorescence microscope. Pictures were taken by a digital camera and digitalized into a computer using the Adobe Photoshop Program.

Thin sections of 40-80 nm in thickness were collected on Nickel grids, and the sections were incubated for 10 minutes in the Tris buffer containing 1% Cold Water Fish Gelatin to block non-specific binding sites, then the grids were incubated overnight at 0-4℃ in the primary antibodies (1:200). After rinsing in the Buffer, a 5 or 10 nm gold conjugated anti-rabbit secondary antibody was applied for one hour at room temperature, the grids were rinsed in buffer and stained for 6 minutes in 2% aqueous uranyl acetate, rinsed and observed with a Zeiss 10C/10 CR electron microscope (Zeiss, Jena, Germany). In control sections, the primary antibodies were omitted or pre-absorbed for 24 hours at 4℃ mixing 3:1 the solution containing the antigen with the purified antibody solution. The grids containing the immunolabeled sections were completely scanned checking for the localization of gold particles in all cell types present in the tissues.

The tissues of the skin, dermis and epidermis, in the normal tail and the mesenchyme and epidermis present in the regenerating tail were studied under the light microscope. The regenerating tail was formed by a loose mesenchyme at the tip, called the blastema, in contact with a multilayered epidermis, the wound epidermis (Fig. 2A). Although most cells were mesenchymal, sparse blood cells and phagocytes were present in the blastema, and these cells were also localized at the base and sparsely within the wound epidermis (Fig. 2B). The electron microscope study allowed a conclusive identification of cells sparsely located among the keratinocytes of the wound epidermis (arrows in Fig. 2B) as granulocytes (see later description).

The study under the fluorescent microscope of sections of normal and regenerating tail after immunostaining with the Ac-BD-27 antibody showed very few and sparse immunofluorescent cells, more frequently encountered in regenerating then in normal tail tissues. In sections of the blood in normal or regenerating condition, sparse granular cells were also seen (see ultrastructural description). Immunofluorescent cells appeared isolated or, more occasionally, forming small clusters in the inter-muscular connectives, dermis, and especially in the regenerating tail connectives and in the apical blastema, and their cytoplasm contained immunofluorescent granules (Fig. 2C, D). Few immunolabeled cells were also seen within the wound epidermis and also in the epidermis of the regenerating tail during early stages of regeneration (Fig. 2E, F). The nature of these granular cells was better defined using the electron microscope.

The systematic observation of the entire skin and the muscle tissues located underneath the dermis in all the grids examined under the electron microscope, including the inter-muscle connective tissue with the sparse blood vessels, confirmed that few cells containing medium-dense cytoplasmic granules were immunopositive (Fig. 3A-C). No labeling was present in the dark and ovoidal granules of the frequent melanophores encountered in the dermis and subdermal connectives.

The cells containing variably immunolabeled granules, both present in the isolated blood sections, or within or outside blood vessels in connective tissues and among the mesenchymal blastema showed a relatively smooth surface, and were mainly identified as heterophilic granulocytes (Fig. 3A). Their heterochromatic nucleus was polymorphic and two or more nuclear sections were often seen in these cells. Granules of larger and denser type, probably corresponding to primary (azurophilic) granules of mammalian granulocytes, and other smaller granules with less electron-density, corresponding to the specific granules of mammalian granulocytes, were observed in these cells. The immunoreactivity for Ac-BD-27 indicated that most frequently labeled were the denser and larger granules of heterophil granulocytes while the remaining cytoplasm was immunonegative (Fig. 3B). Other sparsely labeled dense granules showed a reticulate background (Fig. 3C).

Both the cytoplasm and the variably large granules observed in granulocytes from the blood pellets were paler then in granulocytes present among tissues, suggesting a partial extraction of cell components during preparation of the blood pellets. The content of the immunolabeled granules varied from a fine filamentous meshwork (Fig. 3D) to broad pale areas apparently empty with no gold labeling (Fig. 3E). Controls showed no labeling in both cells and granules of granulocytes (Fig. 3F). Small granules, likely corresponding to the specific granules of mammalian granulocytes, were generally unlabeled. Also the rare basophilic granulocytes encountered in tissues or isolated in the blood pellet contained numerous large granules (over 0.5 µm), some of which appeared weakly labeled (data not shown).

Although the normal epidermis did not contain granulocytes, during wounding and regeneration of the tail, sparse heterophilic granulocytes were seen in the basal layers or among suprabasal keratinocytes in apical areas of the wound epidermis (Figs. 2B, 4A). Among their numerous granules, some granules showed a low (few sparse gold particles) to a higher immunolabeling (more numerous and evenly distributed gold particles) (Fig. 4B). Rare granulocytes also possessed heterogenous granules containing a dense core and a paler halo suggesting they belong to the eosinophilic type (Fig. 4C). Sparse granulocytes with labeled large and dense granules were also observed in few instances within the multilayered regenerating (wound) epidermis of the more apical region of the regenerating tail (Fig. 5A, B). Occasional cytoplasmic labeling was seen in these cells although most labeling remained inside the granules.