Phosphate buffered saline pbs

Immunofluorescence was performed as described in our previous studies.⁴³ (link)⁴⁵ (link)⁴⁸ (link) Briefly, the sections were deparaffinized in xylene and rehydrated through a graded ethanol series. For antigen retrieval, sections immersed in saline sodium citrate buffer were microwave heated for 10 min three times. After cooled to room temperature, the sections were washed by 1×phosphate-buffered saline (PBS; Solarbio), and blocked with 5% donkey serum (Millipore) in 1×PBST (0.3% Triton-X 100 in PBS) at 37°C for 60 min. The commercial antibodies listed in Table S1 were diluted in 3% donkey serum) in 1×PBST (0.2% Triton-X 100) and incubated overnight at 4°C. The sections were then washed, and immunoreactivity was detected using Donkey anti-Rabbit IgG Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 or Donkey anti-Mouse IgG Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 555 (1:500; Thermo Fisher Scientific) for 1 h at room temperature. The sections were counterstained with 5 μg/mL 4’,6-diamidino-2-phenylindole (DAPI; Thermo Fisher Scientific) for 10 min at room temperature and washed with 1×PBST (0.2% Triton-X 100) three times. Slides were visualized using an Olympus FluoView 1000 confocal microscope (Olympus).

To determine the interaction of miR-34a-5p and its potential target gene ACSL1, DF1 cells were seeded in 24-well plates and co-transfected with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) in triplicate with 500 ng of the wild-type or mutant-type plasmid and a final concentration of 80 nM miR-34a-5p mimics or miR-34a-5p mimics NC in serum-free medium. At 48 h after transfection, the cells were washed with 1× phosphate-buffered saline (PBS) (Solarbio, Beijing, China) three times and lysed with passive lysis buffer (PLB). Then, the Renilla luciferase and firefly luciferase activities were measured using a Dual-Luciferase^® Reporter Assay System (Promega, Maddison, WI, USA) on a Fluoroskan Ascent FL instrument (Thermo Fisher Scientifc, Shanghai, China). Renilla luciferase activity served as an internal control to normalize firefly luciferase activity.