Alexa fluor 594 conjugated goat anti rabbit igg h l

SGC7901 and BGC823 cell lines were transfected by (miR-4316-mimic, miR-4316-inhibitor and N.C) or (si-VEGF-A, miR-4316-mimic + si-VEGF-A, miR-4316-inhibitor + si-VEGF-A and N.C) for 48 h, the media were removed then followed with 3 washes of 1× PBS. The cells fixed by using paraformaldehyde 4% at room temperature for 15 min. Subsequently, washed by 1× PBS to removed residual para-formaldehyde. Then, permeabilized cells with 0.1% Triton X-100 made in PBS solution for 15 min at room temperature. Then washed three times by 1× PBS 5 min per time. Block solution BSA 5% incubated at 37 °C for 1 h. Incubated in 37 °C for 1 h in primary antibody anti-rabbit diluted in 1× PBS v/v (1:50), then incubated in 4 °C for overnight. And Alexa-Fluor 594-conjugated Goat Anti-Rabbit IgG (H + L) (protein-tech-USA) was added after washed primary antibody three times 5 min per time stored in the dark for 1 h, washed by 1× PBS 3 times one time 5 min. Finally added DAPI (Sigma) for 7 min then washed three times 5 min per time and observed under the fluorescence microscope (Olympus Inc.).

A rabbit polyclonal antibody against hnRNP-F was acquired from Novus Biologicals (Colorado, USA), and rabbit monoclonal antibodies against AKT, p-AKT, PI3K, p-PI3K, FOXO1, and p-FOXO1 were acquired from Cell Signaling Technology (CST) (Massachusetts, USA). An IgG rabbit polyclonal antibody, IgG mouse polyclonal antibody, Alexa Fluor 594-conjugated goat anti-rabbit IgG (H+L) and Alexa Fluor 488-conjugated goat anti-mouse IgG (H+L) were purchased from Proteintech (Guangzou, China). HRP-conjugated goat anti-rabbit IgG (H+L) and HRP-conjugated goat anti-mouse IgG (H+L) antibodies and antibodies against glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were also purchased from Proteintech (Guangzou, China).