Axio imager z2 optical microscope

Fluorescent immunocytochemistry of the proliferation marker Ki67 was used to estimate the proliferation index between LNCaP cells knocked down for STEAP1 and LNCaP-WT, both treated with the three drugs. LNCaP cells were fixed with 4% paraformaldehyde (PFA) for 10 min at room temperature and permeabilized with 0.1% Triton X-100 for 5 min also at RT. A blocking step was performed by incubating cells with 20% FBS in phosphate buffer saline (PBS) containing 0.1% Tween-20 (PBST) for 1 h at RT and then cells were incubated with rabbit anti-Ki-67 (1:50; cat. no. 16667; Abcam) for 90 min at RT. The Alexa Fluor 546 goat anti-rabbit IgG (1:1,000; Invitrogen; Thermo Fisher Scientific, Inc.) was used as a secondary antibody. This incubation was performed at RT for 60 min. The specificity of the staining was assessed by omission of the primary antibody. Cell nuclei were stained with Hoechst 33342 (5 µg/ml; Invitrogen; Thermo Fisher Scientific, Inc.) for 5 min at RT. Coverslips were washed and mounted onto microscope slides with Dako fluorescent mounting medium (Dako; Agilent Technologies, Inc.). Images were acquired using a AxioImager Z2 optical microscope (Carl Zeiss AG). Proliferation was determined by the percentage of Ki-67-positive cells out of the total number of Hoechst-stained nuclei in eight randomly selected fields per microscope cover glass.