Retiga ex ccd camera

VCaP cells were cultured on poly-L-lysine-coated glass coverslips (BD Bioscience) in androgen-deprived medium for 2 days. Cells were induced with 0.1 nmol/L R1881 and cultured for another 48 hours. Cells were fixed with 4% paraformaldehdye buffered in PBS, permeabilized with 0.1% Triton X-100 in PBS, and blocked with 1% normal horse serum (Vector Laboratories) before incubating with purified IgG. Secondary antibody (Alexa Fluor-488 goat anti-human; Invitrogen) was subsequently applied together with DAPI (40, 6-diamidino-2-phenylindole). F-actin was stained with Alexa Fluor-594 phalloidin (Invitrogen). ERG MAb 9FY was used as a positive control and stained with Alexa Fluor-488 goat anti-mouse secondary antibody. Images were captured using a 40x/0.65 N-Plan objective on a Leica DMIRE2 upright microscope with a QImaging Retiga-EX CCD camera controlled by OpenLab software (PerkinElmer), converted into color, and merged by using Adobe Photoshop.

Tissues were fixed in formalin and paraffin-embedded. Sections (5 μm) were stained with hematoxylin and eosin (H&E) or periodic acid-Schiff (PAS). Slides were analyzed on a Zeiss Axioplan2 microscope and images captured using a Qimaging Retiga EX CCD camera and Openlab 4.0.4 software (PerkinElmer). For immunofluorescence, 5 μm sections of paraformaldehyde-fixed, paraffin-embedded tissues were incubated with anti-Ki67 (SP6 clone, Sigma)) followed by Alexa568-conjugated goat-anti-rabbit and DAPI.
IEC turnover was calculated by Ki67+ cells / total cells (DAPI+) per crypt X 100. Of note, crypt length was under all conditions equally associated with total cells per crypt.