Yu et al. Journal of Experimental & Clinical Cancer Research 2010, 29:107 http://www.jeccr.com/content/29/1/107 Page 2 of 7 of chemotherapy in breast cancer is not ideal. An important reason for this is that breast cancer cells to chemotherapeutic agents are neither sensitive nor resis- tant. Currently looking for the target which could fore- cast the effect of chemotherapy on breast cancer are largely needed. The EADM, 5-Fu, NVB, DDP are the widely-used first-line chemotherapy drugs for breast cancer in the world. In this study MTT assay was used to analyze the relative inhibition effect of four kinds of chemotherapy drugs which include EADM, 5-Fu, NVB and DDP on breast cancer cells, and the relationship between the expression of BCL-2, BAD and the chemosensitivity. Materials and methods Materials 1.1.1 We collected 80 samples of breast carcinoma dur- ing 1998-2002, originated from The First Affiliated Hos- pital of Chongqing Medical University. Including 40 youth breast carcinoma tissuses(age < 35 years old), 40 menopause breast carcinoma tissuses(age > 60 years old);11 cases of clinical Stage I, 47 cases of clinical stage II, 19 patients with clinical stage III, 3 patients with clin- ical stage IV; histological grade I of 26 cases, 46 cases of grade II, III is 8 cases. 10 samples from patients of breast fibroadenoma.10 normal breast tissue samples from 10 patients of side tissue of fibroadenoma. All the samples were made into 5 μm tissue sections 1.1.2 We collected 20 fresh samples of breast cancer, which diagnosed by pathology, without preoperative radiotherapy and chemotherapy, originated from The First Affiliated Hospital of Chongqing Medical Univer- sity. We selected the samples according to the asepsis operation and avoid the necrosis region. One part of the tumor specimens was resected from the primary lesions and transported to our laboratory as quick as possible in RPMI 1640. The other part was put in formal in fixa- tion, dehydration and paraffin imbedding. 1..1.3 Reagent Rabbit anti-human multiclonal BCL-2 antibody and Rabbit anti-human multiclonal Bad antibody were pur- chased from Boshide Bio-engineering company. Epirubi- cin, Fluorouracil, Navelbine and Cisplatin were dissolved in the mother liquor separately by physiological saline, and then disposed the mother liquor into fluid (100 × PPC), positive pressure filtration sterilization, -20°C preservation. 1.2.1 Immunohistochemistry Immunohistochemistry was carried out on 5 μm tissue sections from paraffin blocks using the avidin-biotin immunoperoxidase method, The following antibodies were used: Rabbit anti-human multiclonal BCL-2 anti- body and Rabbit anti-human multiclonal Bad antibody. Briefly, the paraffin sections were deparaffinized with xylene and rehydrated through a series of descending graded ethanol. Endogenous peroxidase activity was blocked by incubation for 15 min in 0.3% H2O2 buffer. To unmask the epitopes of BCL-2 and BAD microwave- processing pretreatment was carried out in a citrate buffer, pH = 6.0 for 10 min.. Subsequently, Rabbit anti- human multiclonal BCL-2 antibody or Rabbit anti- human multiclonal BAD antibody were applied. Biotinylated secondary antibody and avidin-biotin- complex with horseradish peroxidase were applied, fol- lowed by the addition of the chromogen. Finally, slides were counterstained with hematoxylin, dehydrated in ascending ethanol, cleared with xylene, and mounted with coverslips using a permanent mounting medium. Result: According to the percentage of the dyeing posi- tive cells(A), The dyeing positive cell number of zero is 0, <30% is 1, 30%~60% is 2, >60% is 3. According to the dyeing intensity (B), the achromatic color is 0, the weak dyeing is 1, the dyeing is 2, the strong dyeing is 3; The total score (A + B) ≥ 3 divides into the positive expres- sion, <3 divides into the negative expression. Immuno- histochemical results to determine criterion-referenced method of Shimizu [1]. 1.2.2 Cell separation, Cell Culture and MTT assay We adopt mechanical method obtained unicell suspen- sion. First, washed the specimens with normal saline (including penicillin 300 μ/ml streptomycin 300 μ/ml) repeatedly to remove necrotic tissue and blood clots, put in the aseptic plate, then adding them into a little culture medium, used eye scissors cut the specimens into paste, 200 Stainless steel wire grit of 200 mesh screen was cell suspension, it was obtained by filtering the minced tissue, though a stainless steel wire grit of 200 mesh screen, checked for the viability and counted, then centrifuge in 1000 r/min, 10 min; regulated the cell concentration into 5 × 104 /l by RPMI1640(containing fetal calf serum, penicillin 100 μ/ml streptomycin 100 μ/ ml), vaccinated the cell in 96-well microtiter plates, 180 μl per well; Each well joined chemotherapeutic agent 20 μl separately (drug level: 10 × PPC, 1 × PPC, 0.1 × PPC), each level set up 3 duplicate holes; Simulta- neously set up the cell control group and the blank con- trol group. Then, the plates were incubated at 37°C in a humidi- fied atmosphere containing 5% CO2 for 48 h. After incubation, 20 μl MTT solution was added to each well, and the plate was incubated for 4 h again; Added DMSO 200 μl, The absorbance of each well was deter- mined in the same manner as used in the MTT assay. The inhibition rate was calculated by using the formula inhibition rate(IR, %) = (1-mean absorbance of the treated wells/mean absorbance of the control wells) × 100%.