Rnasezap

Twenty week old TRAMP mice and non-transgenic littermates were euthanized, tissues were harvested in an RNase-free manner. Incisions were made in the anterior abdomen with RNase-Zap (Ambion, Austin, TX) treated forceps and scissors. The vas deferens, ureters, and any connective tissue were clipped away from the genitourinary (GU) complex. The entire GU complex consisting of the bladder, seminal vesicles and the prostate along with the urethra was removed from the abdominal cavity and placed in cold RNase-free PBS (Ambion) in an RNase-Zap treated petri-dish and micro-dissected to excise the dorsolateral prostate, either snap frozen in liquid nitrogen and stored at −80°C until RNA isolation or used immediately for RNA isolation. Small sections of the prostates from TRAMP mice and non-transgenic littermates were preserved in buffered formalin. Samples were stained with hematoxylin and eosin for histological analysis.

A FACSARIA IIu (BD Biosciences) was used for cell sorting. Prior to each sorting run, all parts of the sort chamber were cleaned with RNaseZap (Life Technologies), and the inlet tube and flow cell were cleaned with 10% Contrad70 followed by RNasin/PBS. Dead cells were excluded based on TO-PRO-3 staining [40 ]. Blue and red lasers were used for excitation. A live gate was set using a FSC-A versus TO-PRO-3 fluorescence dot plot. A cut-off of 3% living TO-PRO-3⁻ cells was used to exclude samples from further analysis (7 samples exceeded this cut-off and were excluded). Cell doublets were excluded using FSC-H versus FSC-W and SSC-H versus SSC-W pulse-processing. Single-stained samples were used to correct for spectral overlap. EpCAM⁺ and CD45⁺ single-positive cells in the double-stained samples were sorted using a 16,16 purity mode, a 100-μm nozzle at 20 psi and a drop frequency of 30.8 kHz. A representative example of the restricted gating strategy for sorting pure EpCAM⁺ tumor epithelial cells and CD45⁺ immune cells is shown in Figure 1. Cells were collected in 2-ml tubes containing 1 ml ice-cold RNAprotect Cell Reagent (Qiagen, Hilden, Germany), mixed and kept on ice until RNA isolation.

For LCM analysis, each sample was handled individually. All room temperature equipment was cleaned using RNAse Zap (Life Technologies, CA, USA), and cryotome with 70% EtOH prior to procedures. Samples were removed from liquid N₂ while kept inside cooled cryotome chamber, and directly mounted in Tissue-Tek O.C.T. compound embedding medium (Sakura Finetek, CA, USA). The mounted sample was left to equilibrate inside the cryotome for 30 min. Cryosections (10 μm) were made using a Leica CM3050 S (Leica, Wetzlar, Germany) and directly placed on a pre-chilled LCM membrane slide. When the membrane slide was filled with sections, it was briefly exposed to room temperature in order to let sections adhere to the membrane surface, and subsequently placed in 100% EtOH (30 sec). The following staining protocol was modified from the Histogene LCM staining kit (Cat. No. KIT0401, Life Technologies, CA, USA). Sections were washed twice (5 sec) with dH2O with added ProtectRNA RNase inhibitor (Cat. No. R7397, Sigma-Alrich, MO, USA) followed by staining with Histogene staining solution. After subsequent washing steps (3 x 30 sec 100% EtOH, 2 x 2 min Xylene, 1 x 5 min Xylene) sections were air dried (30 sec) and placed in desiccator (30 min). After drying, the membrane slide was processed in an LCM microscope (MMI CellCut on Olympus IX71, Eching, Germany).