Phb1 rabbit antibody

LNCaP cells (50 000 cells) were seeded in a round disc and incubated in 2 mL of RPMI1640 medium containing 10% FBS for 24 h. After PHB1 silencing with PHB1 siRNA-loaded NPs or ACUPA-NPs, the cells were fixed with 4% paraformaldehyde. The cells were then permeabilized by incubation in 0.2% Triton X-100 in PBS for 5 min, followed by washing with PBS (3 × 5 min). Thereafter, the cells were blocked with blocking buffer (2% normal goat serum, 2% BSA, and 0.2% gelatin in PBS) at room temperature for 1 h. After washing the cells with PBS (3 × 5 min), PHB1 rabbit antibody (Abcam) diluted in 1% BSA solution was added and the cells were incubated for 1 h. Subsequently, PBS buffer (3 × 5 min) was added to the cells, which were then further incubated with Alex Fluoro 647-linked secondary antibody and Alex Fluoro 488-conjugated phalloidin for another 1 h. After washing with PBS buffer (3 × 5 min), the cells were stained with Hoechst 33342 and finally viewed under a FV1000 CLSM (Olympus).

Immunohistochemistry staining was performed on formalin-fixed paraffin-embedded tumor sections. Briefly, tumor slides were first heated to 60 °C for 1 h, desparaffinized with xylene (3 × 5 min), and washed with different concentrations of alcohol. After retrieval of antigen using DAKO target retrieval solution at 95–99 °C for 40 min, followed by washing, the slides were blocked with peroxidase blocking buffer (DAKO Company) for 5 min. After washing the buffer (DAKO Company), the slides were incubated with PHB1 rabbit antibody (Abcam) diluted in DAKO antibody solution for 1 h. The slides were then washed and incubated with peroxidase-labeled polymer for 30 min. After washing and staining with DAB + substrate-chromogen solution and hematoxylin, the slides we remounted and viewed under an MVX10 MacroView dissecting scope equipped with an OlympusDP80 camera.