Ben Jemaa et al. Journal of Experimental & Clinical Cancer Research 2010, 29:171 http://www.jeccr.com/content/29/1/171 Page 3 of 9 washed in de-ionized water. To retrieve antigens, sec- tions were incubated in citric acid solution (0.1 M, pH 6) for 20 minutes in 98°C using a water bath. Slides were allowed to cool for another 20 min, followed by washing in de-ionized water. Endogenous peroxidase activity was quenched by incubation with Peroxidase Block for 5 minutes. Each incubation step was carried out at room temperature and was followed by two sequential washes (5 min each) in TBS. Sections were incubated with Protein Block for 5 minutes to prevent non-specific binding of the first antibody. Thereafter, the primary antibodies were applied at a dilution of 1/ 50 (PSMA) and 1/100 (PSA, CD34) in antibody diluents (Dako, Glostrup, Denmark) at room temperature for 30 minutes. Afterwards, the sections were incubated with Post Primary Block for 30 minutes to block non-specific polymer binding. The sections were incubated with NovoLink™Polymer for 30 minutes followed by incuba- tions with 3, 3’-diaminobenzidine (DAB) working solu- tion for 5 minutes to develop peroxidase activity. Slides were counterstained with hematoxylin and mounted. Stainig specificity was checked using negative controls. Prostatic tissues of each type were incubated in blocking peptides (Santa Cruz Biotechnology, Santa Cruz, CA, USA) instead of primary antibodies. A comparative quantification of immunolabeling in all tissues types was performed for each of the three anti- bodies. Of each prostate, six histological sections were selected at random. In each section, the staining inten- sity (optical density) per unit surface area was measured with an automatic image analyzer (Motic Images Advanced version 3.2, Motic China Group Co., China) in 5 light microscopic fields per section, using the ×40 objective. Delimitation of surface areas was carried out manually using the mouse of the image analyzer. For each positive immunostained section, one negative con- trol section (the following in a series of consecutive sec- tions) was also used, and the optic density of this control section was taken away from that of the stained section. From the average values obtained (by the auto- matic image analyzer) for each prostate, the means ± SEM for each prostatic type (normal prostate, BPH and PC) were calculated. The number of sections examined was determined by successive approaches to obtain the minimum number required to reach the lowest SEM. The statistical significance between means of the differ- ent prostate group’s samples was assessed by the Fisher exact test and the one-way ANOVA test at p≤0.05 (GraphPad PRISMA 5.0 computer program). Results We examined human histological specimens (NP, BPH and PC) by immunohistochemistry to evaluate the rela- tionship between the co-expression of prostate- associated antigens (PSMA and PSA) and the degree of vasculariza- tion (intensity of immunoreaction to CD34). We didn’t see any immunoreactivity in the negative controls incubated with blocking peptides (Figure 1A). Immunorectivity for PSMA appeared in 83% of NP, 86% of BPH and 97% of PC samples. In NP and BPH sam- ples, PSMA was exclusively expressed in the cytoplasm of luminal epithelial cells, whereas we found it only expressed in the tumor cells of the PC specimens. We wanted to look at the expression of PSMA in blood vas- cular, we stained adjacent sections with anti-CD34 and anti-PSMA antibodies of our samples and we found that endothelium of both benign and malignant prostate tis- sues were deprived from PSMA expression (Figure 1C, G and 1K). We used Motic advanced software to calculate the optic density (OD) that correlates with the antigen expression. We found that the mean of PSMA expres- sion was significantly increased in benign prostate glands compared with normal prostate tissue (respec- tively 16.14 ± 0.17 and 3.7 ± 0.18) (p = 0.008). The highest level of PSMA expression was found in primary prostate cancer (30.72 ± 0.85) which significantly dif- fered from benign (p < 0.0001) and normal prostatic tis- sue (p < 0.0001) (Figure 2A). Unlike PSMA, PSA expression was found the highest in hyperplastic epithe- lial cells (Figure 2B). Scanty immunoreactivity to PSA was localized in the cytoplasm of epithelial cells in nor- mal prostate (Figure 1D). Figure 2B showed that the intensity of immunoreaction to PSA decreased from BPH samples to prostate adenocarcinoma (34.39 ± 0.53 and 17.85 ± 1.21, respectively) (p < 0.0001). As shown in this figure, 57% of PC samples positive for PSA have a similar PSA expression level distribution to NP sam- ples, whereas 43% have a similar PSA expression level distribution to BPH samples. PSA staining was present in 83% of NP, 75% of BPH samples and 74% of PC samples. To look at the vasculature in our samples, we immu- nostained them with anti-CD34 mouse using IHC method. CD34 consistently showed immunoreactivity in the plasma membrane of endothelial cells in all pros- tates specimens (Figure 1E, I and 1M). Measuring the optical density of CD34 immunostaining, we found that there is a significant difference in vasculature density between normal, hyperplasia and tumors in our collec- tion (Figure 2C). Interestingly, similar to PSMA, CD34 staining was found more abundant in PC specimens (12.08 ± 0.29), compared with NP and BPH (p < 0.0001). Vessel density was higher in BPH compared to NP samples (8 ± 0.11 and 2.34 ± 0.15, respectively) (p < 0.0001) (Figure 2C). To study the relationship between PSMA and PSA expression and microvessel density in BPH and PC