Angiogenesis, or neovascularization, is known to play an important role in the neoplastic progression leading to metastasis. CD31 or Factor VIII-related antigen (F VIII RAg) immunohistochemistry (IHC), is widely used in experimental studies quantifing tumor neovascularization in immunocompromised animal models implanted with transformed human cell lines. Quantification, however, can be affected extensively by variations in the methodology used to measure vascularization including antibody selection, pretreatment with antigen retrieval and evaluation techniques. To examine this further, we examined the microvessel density and the intensity of microvascular staining among five different human tumor xenografts and a mouse syngeneic tumor using anti-CD31 and F VIII RAg IHC staining. Different antigen retrieval methods also were evaluated. Maximal retrieval of CD31 was achieved using 0.5 M Tris (pH 10) buffer, while maximum retrieval of F VIII RAg was achieved using 0.05% pepsin treatment of tissue sections. For each optimized retrieval condition, compared to F VIII RAg, anti-CD31 highlighted small vessels better. Furthermore, the microvessel density of CD31 was significantly greater than that of F VIII RAg decorated vessels (p < 0.001). The choice of antibody and antigen retrieval method has a significant affect on immunohistochemical findings when studying angiogenesis. One also must use caution when comparing studies in the literature that use different techniques and reagents.
(AFC) S U M M A R Y Osteopontin (OPN) is an adhesive, matricellular glycoprotein, whose expression is elevated in many types of cancer and has been shown to facilitate tumorigenesis in vivo. To understand the role of OPN in human skin cancer, this study is designed to determine whether OPN is expressed in premalignant [solar/actinic keratosis (AK)] and in malignant skin lesions such as squamous cell carcinomas (SCC) and basal cell carcinomas (BCC), as well as in normal skin exposed or not exposed to sunlight. Immunohistochemical analyses showed that OPN is expressed in SCC (20/20 cases) and in AK (16/16 cases), which are precursors to SCC, but is absent or minimally expressed in solid BCC (17 cases). However, positive staining for OPN was observed in those BCC that manifest differentiation toward epidermal appendages such as keratotic BCC. In sunlight-exposed normal skin, OPN is minimally expressed in the basal cell layer, but in contrast to those not exposed to sunlight, OPN is more prominent in the spinous cell layer with increasing intensity toward the granular cell layer. Additionally, OPN is expressed in the hair follicles, sebaceous glands, and sweat glands of normal skin. In conclusion, these data suggest that OPN is associated with keratinocyte differentiation and that it is expressed in AK and SCC, which have metastatic potential, but minimally expressed in solid BCC. (J Histochem Cytochem 56:57-66, 2008)
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