(Figure 4a,b). We found a diffuse immunore- activity of Tβ4 in the stroma of adult prostate (Figure 4b), but no reactivity was detected in the foetal prostate stroma (Figure 4a). A fine granular positivity was present in the foetal ovary (Figure 5a), while immunoreactivity of the peptide was homogeneously weak in adult oocytes (Figure 5b). Stroma was negative in Table 1. Immunoreactivity of Tβ4 in differ- ent organs and cells of the human geni- tourinary tract. Foetus Aduld Kidney Ducts Glomeruli Bladder Transitional epithelium Stroma Endometrium Glands Stromal cells Prostate Glands Stroma Isolated cells Ovary Oocyte Testicle Spermatic ducts Interstitial cells Diffuse Negative Focal Negative Negative Focal Negative Negative Focal Focal Negative Focal Diffuse Negative Diffuse Focal Diffuse Diffuse Focal Diffuse Diffuse Diffuse Focal Diffuse Figure 2. Bladder. (a) Foetuses: the developing transitional epithelium shows a weak and dif- fuse cytoplasmic immuoreactivity of Tβ4 (arrow). The surrounding stroma shows a mild diffuse immunoreactivity of the peptide. Scale bar: 50 μm. (b) Adults: perinuclear coarse gran- ules, immunoreactive for Tβ4, are detected in the cytoplasm of transitional epithelium (arrow). The stroma shows a focal positivity of the peptide (arrowhead). Scale bar: 50 μm. Original paper samples were fixed in 10% formalin, routinely processed and paraffin-embedded. Immuno- histochemistry was performed on 5 μm-thick sections, using the labelled streptavidin-biotin complex system (LSAB2, Dako) in a Dako Autostainer (DakoCytomation, Carpintera, CA, USA). Heat-induced antigen retrieval was car- ried out by steaming unstained sections in Target Retrieval Solution (Dako TRS pH 6.1) for 30 min. Tissue sections were incubated (30 min at room temperature) with the monoclon- al anti-thymosin β4 antibody (Bachem, Bubendorf, Switzerland). Sections of a reac- tive human adult lymph node with activated macrophages were used as positive controls.22 As a negative control the same procedure was applied omitting the primary antibody. Ethics statements The study protocol and written consent forms were approved by the Ethics Human Studies Committee of University Medical Centre of Cagliari (according to the instructions of the Declaration of Helsinki). Full written consent forms were obtained from the parents of the newborns and all rules were respected. For the specimens from adults, we obtained written consent from their next of kin. Results The immunostaining for Tβ4 appeared homogeneous or granular and was always restricted to the cytoplasm of positive cells. No nuclear reactivity was observed in this study. No significant differences were found in the immunohistochemical pattern for Tβ4 among the four foetuses analyzed, as well as the four adults observed in this study (Table 1). The immunoreactivity for Tβ4 in foetal kidneys par- alleled the immunoreactivity in the adult. A weak diffuse cytoplasmic positivity was detect- ed in developmental and mature ducts while the glomeruli were constantly negative (Figure 1a,b). In the bladder, a weak immunoreactivity of the peptide was observed in the immature transitional epithelium, which changed to a coarse granular reactivity in the mature epithelium of the adult bladder (Figure 2a,b). The surrounding stroma showed mild diffuse immunoreactivity of Tβ4 in the foetus and focal in adults (Figure 2b). No immunoreactivity for Tβ4 was observed in foetal endometrial glands. Only scattered stromal cells showed a weak cytoplasmic immunoreactivity (Figure 3a). A mild, granular cytoplasmic immunoreactivity of Tβ4 was present in the endometrial glands of adults, while the absence of any significant immunoreactivity was found in the stroma (Figure 3b). No reactivity for Tβ4 was detected in the foetal or adult prostatic gland epithelium Figure 1. Kidney. (a) Foetuses: a diffuse cytoplasmic immunoreactivity of Tβ4 is present in developing ducts (arrow). The immature glomuruli are negative (arrow- head). Scale bar: 25 μm. (b) Adults: pri- mary and secondary ducts show a diffuse cytoplasmic immunoreactivity of Tβ (arrow). The glomeruli are negative (arrowhead). Scale bar: 25 μm. Figure 3. Endometrium. (a) Foetuses: no immunoreactivity of Tβ4 is observed in devel- oping endometrial glands (arrow). Only scat- tered stromal cells show a weak cytoplasmic immunoreactivity (arrowhead). Scale bar: 100 μm. Inset: scattered stromal cells with a cyto- plasmic immunoreactivity (arrows). Scale bar: 25 μm. (b) Adults: a mild granular cytoplasmic immunoreactivity of Tβ4 is observed in the endometrial glands (arrow) while the surround- ing stroma has no significant immunoreactivity of the peptide. Scale bar: 25 μm. [page 202] [European Journal of Histochemistry 2010; 54:e43]