Mouse α psma

Crude cell lysates were prepared from cells using 100 µL RIPA buffer, 2 µL phenylmethylsulfonyl fluoride (1 mM), and 100 µL protease inhibitor cocktail (stock: 1 tablet/5 mL water). Protein concentrations were determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, USA/MA). A total of 30 µg of protein was loaded in each well on an Any kD™ Mini-PROTEAN® TGX™ Precast Protein Gel (Bio-Rad, USA/CA) and blotted onto a polyvinylidene fluoride (PVDF) membrane. Unspecific signal was blocked with 5% milk powder in TBST. The membranes were incubated overnight at 4 °C with primary antibodies: mouse α-PSMA (Abcam, 1:1000) or rabbit α-GAPDH (Cell Signaling, 1:2000). After washing, the secondary antibody horse α-mouse IgG HRP (Cell Signaling, 1:1000) or goat α-rabbit IgG HRP (Cell Signaling, 1:1000) was applied for 1 h at room temperature. The membrane was prepared for signal detection with Pierce™ ECL Western Blotting Substrate (Thermo Fisher Scientific, USA/MA) and subsequently analyzed with an Image Quant LAS 4000 Luminescent Analyzer (GE Healthcare, USA/IL).

The inserts were discarded, and HUVECs were washed with PBS, fixed with 4% PFA for 15 min, and permeabilized with 0.1% triton/PBS. Unspecific signal was blocked with 5% goat serum. Samples were stained overnight at 4 °C with primary rabbit α-cluster of differentiation 31 (CD31) (Invitrogen, 1:100) and mouse α-PSMA (Abcam, UK, 1:250) antibodies. As secondary antibodies, a goat α-mouse dylight 488 (Abcam, UK, 1:1000) and a goat α-rabbit AF 555 (Cell Signaling Technology, USA/MA, 1:1000) were applied for 1 h at room temperature. DAPI staining was applied for 3 min, and the cells were mounted with Mowiol. Images were acquired with a Zeiss Imager Z.1 microscope (Zeiss, Germany).

Spheroids were fixed with 4% PFA for 15 min, embedded in Tissue-Tek O.C.T. (Sakura Finetek, Germany), and snap-frozen in cold 2-methylbutan (Carl Roth, Germany). Cryosections (10 µm) were obtained using a Leica JUNG CM3000 cryostate (Leica Microsystems, Germany) and transferred on SuperFrost slides (Thermo Fisher Scientific, USA/MA). Sections were immersed in PBS, permeabilized with 0.1% triton/PBS, and blocked with 5% goat serum. Samples were stained overnight at 4 °C with primary rabbit α-CD31 (Invitrogen, USA/MA, 1:100) and mouse α-PSMA (Abcam, UK, 1:250) antibodies. After washing, sections were incubated with 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 staining was applied for 3 min and the cells were mounted with Mowiol. Images were acquired with a Zeiss Imager Z.1 microscope (Zeiss, Germany).