Goat α mouse alexa fluor 488

Sections were immersed with PBS, permeabilized for 5 min with 0.1% triton/PBS, and blocked for 90 min with 5% goat serum at room temperature. Samples were incubated overnight at 4 °C with primary mouse α-PSMA (Abcam, UK, 1:250) and rabbit α-CD31 (Invitrogen, USA/MA, 1:100), or rabbit α-HIF1α (Novus biologicals, USA/CO, 1:100) antibodies. After washing 3 × with PBS, sections were incubated with goat α-mouse Alexa Fluor 488 (Thermo Fisher Scientific, USA/MA, 1:1000) and goat α-rabbit Alexa Fluor 555 (Cell Signaling, USA/MA, 1:1000) antibodies for 1 h at room temperature. DAPI (Merck, Germany) staining was applied for 3 min and the cells were mounted with mowiol. Images were acquired with an Imager Z.1 microscope (Zeiss, Germany).

Cells were seeded at low density in 24-well plates on round coverslips coated with Poly-L-lysine solution (Sigma). For staining, cells were fixed for 10 min in 4% PFA (at RT) and subsequently washed thoroughly with PBS. Cells were blocked in blocking solution (PBS, 0.1% Tween-20, 5% BSA) for 1 h at RT, followed by incubation with primary antibody in blocking solution (1:100, 1 h at RT). Then, cells were washed three times for 5 min with PBS-T, incubated in PBS-T with secondary antibodies (all Thermo Scientific) goat α-mouse Alexa Fluor 488 (#A-11001), goat α-mouse Alexa Fluor 647 (#A-21235), goat α-rabbit Alexa Fluor 568 (#A-11011), goat α-rabbit Alexa Fluor 647 (#A-21244) at 1:1000 dilution for 1 h at RT and washed three times with PBS-T. After nuclear counterstaining with DAPI in PBS-T, coverslips were washed in PBS and mounted on glass slides using mounting medium (2.5% 1,4-Diazabicyclo-octane, 10% Mowiol 4-88, 25% glycerol and 0.1 M Tris-HCl). Images were acquired using a Leica SP5 confocal laser scanning microscope (×63 magnification). Images were analyzed using the Fiji/ImageJ software^{38 (link)}. The Pearson’s correlation between the respective channels was quantified with the Coloc2 tool of the Fiji software using the auto-threshold function.

Infected cells were fixed in 4% PFA and washed two times with a wash buffer consisting of PBS containing 0.2% BSA and 0.2% Triton X-100, and then incubated for 1 h in blocking buffer composed of 2% BSA and 0.2% Triton X-100 in PBS. Cells were next incubated in 2 μg 4G2 antibody/ml wash buffer for 1 h, washed three times with additional wash buffer, and incubated 1 h with 1 μg goat α-mouse Alexa Fluor 488 (Thermo Fisher)/ml wash buffer. Cells were washed several times, with one wash containing 4′,6-diamidino-2-phenylindole (DAPI; Thermo Fisher) to stain cell nuclei, and visualized by fluorescent microscopy.