Min in a citric Acid buffer. The antigen-antibody Body complex was diaminobenzidine with 3.3-L Solution and visualized with AUY922 gegengef matoxylin H Rbt. As a negative control, normal rabbit IgG was used instead of primary body Ren Antique. TACC2 Immunreaktivit t was detected in the cytoplasm, and was Immunreaktivit t evaluated in more than 1000 carcinoma cells for each case. Subsequently End, the percentage of Immunreaktivit t determined by two pathologists. F ll With more than 10% of TACC2 Immunreaktivit t were not considered positive TACC2 prostate cancer in this study. TACC2 a correlation between clinicopathological factors and Immunreaktivit t was determined using a student of St-test, a cross table with the 2 test, or correlation coefficient and regression equation. Cancer-specific survival and PSA st Destructive survival curves were generated using Kaplan-Meier method, and statistical significance was based on the log-rank test. K Kingdom and multivariate analyzes were proportional hazards model using PROC PHREG with Cox in SAS software. In order to synchronize immunocytochemistry the LNCaP cells, the cells with 10% serum after serum starvation were stimulated for 24 h. After incubation for 24 h, the cells were washed three times with PBS and fixed in 4% formalin in PBS for 15 min at room temperature. Min after a further washing step with PBS and blocking in 5% normal goat serum / PBS for 30 were initially the cells First with monoclonal mouse MPC-3100 anti-tubulin and polyclonal rabbit anti-TACC2 with in 5% normal goat serum / PBS overnight, three times PBS and then incubated with fluorescein isothiocyanate-conjugated anti-rabbit IgG and rhodamine-conjugated anti-mouse IgG antibody body incubated for 1 h.
Nuclei were stained with 4,6 diamidino 2 phenylindole Rbt. Deckgl These were mounted in glycerol and visualized. We compared the proportion of mitosis in cells transfected siRNA contr The LNCaP cells with siTACC2 with double-sided 2 test transfected. Feeder We interviewed five Llige fields and determine whether cells in mitosis or premetaphase postmetaphase. Cell cycle analysis for cell cycle analysis unsynchronized, the cells were harvested after 96 h of incubation after transfection of siControl and siTACC2. For synchronization in G0/G1, serum starvation was for24 h using phenol red-free RPMI 1640, after which the cells with f Fetal K were 20% calf serum treated. For synchronization in the G2 / M, we have nocodazole for 14 h, after Ritonavir which the medium was changed. After incubation for 24 h, the cells were centrifuged, washed in PBS and then fixed by slow addition of 3 ml of 70% ice cold ethanol soft stirring. They were stored at 4 ° C until use. The tag analysis the cells were centrifuged, washed in PBS, in 1 ml PBS containing 100 g / ml ribonuclease with 106 cells, and incubated at 37 ° C for 30 min. To determine DNA content, 30,000 cells by flow cytometry FACS Calibur were analyzed by CellQuest software. Western blot of whole cell lysates for the analysis were performed using a lysis buffer. The lysates were resolved by SDS-PAGE St and 8% electroblotted onto Immobilon P transfer membrane. The membranes were first First with prime Rem Antique Body and anti-rabbit peroxidase-conjugated anti-mouse IgG antibody Body or incubated for 1 h. After extensive washing, the antibody antigens.