Int J Colorectal Dis (2010) 25:1231–1242 primary antibody incubation for half an hour and secondary antibody incubation for 15 minutes, DAB detection kit was applied according to the manufacturer's guidelines. All sections were counterstained with haematoxylin and blueing reagent. Dehydration in ethanol and xylene series was carried out, and finally the sections were mounted permanently by a cytosol embedding medium (Microm, Walldorf, Germany). Negative controls with- out primary antibody did not show antibody-induced labelling. In a second part of the study, tumour blocks of 17 patients (mean age 74 years, range 57–86 years) with ultralow rectal cancer (pT2, N0, M0, GII–pT3, N1, M0, GIII) following abdominoperineal resection or anterior resection of the rectum without preoperative radiochemo- therapy and resection specimens of four patients (mean age 73, range 62–82 years) with anal squamous cell carcinomas with preoperative radiochemotherapy were processed accordingly. The samples were obtained from the Institute of Pathology, Innsbruck. Results The following subdivision is equivalent to the nomen- clature and subdivision of the anorectum proposed by Fenger [4]: The rectal zone (RZ) is characterized by regularly arranged colonic crypts (Fig. 1). The anorectal zone (ARZ) is a continuation of the rectal mucosa with shortening and irregularity of the crypts (Fig. 1). The anal Fig. 1 Schematic drawing of the anorectum and its mucosa 1233 transitional zone (ATZ) is characterized by anal columns and sinuses and has epithelial variants (columnar, cuboidal and squamous cells) (Fig. 1), which in most cases end at the dentate line (DL). The length of both, ARZ and ATZ, is variable. The squamous zone (SZ) is covered with uninterrupted squamous epithelium (Fig. 1) and gradually merges into the perianal skin (PS), covered by keratinized squamous epithelium (Fig. 1). Nine- to eleven-week-old fetuses Rectal zone Whereas the rectum showed a stratified columnar and regularly arranged epithelium without goblet cells in 8-week-old embryos [10], protuberances of the epithelium and the propria, that is, first signs of transitional villi, as well as goblet cells appeared in 9/10-week-old fetuses (Fig. 2a). Positive labelling for CK 18 was observed throughout all rectal epithelial cell layers (Fig. 2b). They were negative for CK 7 (Fig. 2c) in the RZ. Whereas negative reaction was found for CK 14 (data not shown), positive staining reaction was demonstrated for CDX2 (Fig. 2d). Some superficial epithelial cells labelled positive for CK 20 (Fig. 2e). Anorectal zone In the ARZ, positive labelling for CK 18 was detected (Fig. 2b, g), and also some isolated positive cells for CK 7 with an accumulation near the transitional zone (Fig. 2c). Anal transitional zone The transitional zone was lined by two- to five-layered stratified cuboidal epithelium (Fig. 2f) with vertical surface cells in the cranial area and more flattened caudally. The differentiation of the anal columns and sinuses already started in 9/10-week-old fetuses. The epithelium of the transitional zone showed strong pan- epithelial reaction for CK 18 (Fig. 2g) as well as for CK 7 (Fig 2h), with pronounced immunoreactivity in the supra- basal layers. In this early stage, CK 14 was not detected in epithelial cells of the transitional zone (data not shown). Negative reaction was found for CK 20 (data not shown) and CDX2 (Fig 2i). Squamous zone and perianal skin The anal canal was lined by uninterrupted stratified squamous epithelium (Fig. 2j). The superficial cells were