Inverted microscope

Filamentation in liquid medium was assayed at 37°C in RPMI 1640 supplemented with l-glutamine (Cellgro, Manassas, VA) and buffered with 165 mM 3-morpholinopropanesulfonic acid (Sigma, St. Louis, MO) to pH 7.0 (“buffered RPMI 1640”). In brief, buffered RPMI 1640 with or without DOX was inoculated with cells from overnight cultures at a final density of 5 × 10⁶ cells/ml, followed by incubation at 37°C with shaking at 200 rpm. Cells were visualized by DIC microscopy after 24 h by using a Zeiss EC Plan-NeoFluar 63×/1.25× oil objective (Carl Zeiss AG, Jena, Germany).
Filamentation was assayed on solid YPD with 10% (vol/vol) fetal calf serum (FCS), Medium 199 supplemented with l-glutamine (M199), and Spider medium, as described previously (24 (link)). YPD agar was used for embedded colony observation as described previously (32 (link)). All plates were prepared with and without 20 μg/ml doxycycline. Aliquots of 3 μl of cells from overnight cultures were spotted onto agar plates and incubated at 37°C for 24 h. For embedded colony observation, molten YPD agar was cooled to approximately 45°C and cells from overnight cultures were added to a concentration of 20 cells/ml, poured into individual petri dishes, and incubated for 24 h at 30°C. Filamentation was observed with an inverted microscope (Fisher Scientific, Waltham, MA) at 100× and 400× total magnification.

As previously described [17 (link)], terminal tumor tissue was stored in a 10% neutral buffered formalin for fixation and subsequent paraffin embedment. Formalin-fixed paraffin-embedded tumors were sectioned and stained with hematoxylin (Vector Laboratories, Newark, CA, USA, H-3401) and eosin (Harleco, Darmstadt, Germany, 200-12) to evaluate tissue morphology. Images were taken using an inverted microscope (Fisherbrand, Waltham, MA, USA, 03-000-013) with a digital camera attachment (Amscope, Irvine, CA, USA, MU503) at 40× magnification. Scale bars: 50 µm.