Alexa fluor 488 conjugated fluorescein labeled anti rabbit secondary antibody

For immunostaining, PC3 cells (1.5 × 10⁵) were grown on coverslips in 6-well plates and transfected with custom Cy3-labeled NC or miR-214 mimic (Bioneer, Oakland, CA) for 48 h. Cells were washed with PBS, fixed with 4% paraformaldehyde for 15 min, then cells were blocked with 2% bovine serum albumin for 1 h and incubated with anti-N-Cadherin, and anti-E-Cadherin rabbit primary antibodies (diluted 1:200) at 4 °C for 18 h. For PTK6 staining, transfected cells were fixed and permeabilized with 0.25% Triton X-100 for 15 min and cells were blocked and incubated with anti-PTK6 rabbit primary antibody (diluted 1:200) at 4 °C for 18 h. Cells were washed twice with PBS and incubated with Alexa-Fluor® 488-conjugated fluorescein-labeled anti-rabbit secondary antibody (Invitrogen). Following immunostaining, cells were mounted with Vectashield mounting medium (Vector Laboratories, Burlingame, CA) containing a nuclear DAPI (4′,6-diamidino-2-phenylindole) stain. Images were captured by confocal microscopy using a ZEISS LS 800 microscope.

HaCaT cells and skin cancer cell lines A431 and A2058 (1.5 × 10⁵) were grown on coverslips in 6-well plates and transfected with custom made Cy3-labeled NC or miR-205-5p mimic (Bioneer, Oakland, CA) for 48 h. After washing with PBS, cells were fixed with paraformaldehyde (4%) for 15 min, permeabilized with 0.25% Triton X-100 in PBS, and blocked with 2% bovine serum albumin in PBS for 1 h at room temperature. Cells were washed with PBS two times (15 min each) and incubated with anti-TNFAIP8 or anti-LB3β I/II rabbit primary antibodies (diluted 1:500) at 4 °C for 18 h. Cells were washed again twice with PBS and incubated with Alexa-Fluor 488-conjugated fluorescein-labeled anti-rabbit secondary antibody (Invitrogen). After the immunostaining, cells were washed three times with PBS mounted with Vectashield-DAPI mounting medium (Vector Laboratories, Burlingame, CA, Cat # H-1500-10). Slides were observed under a confocal microscope ZEISS LS 800 and images were captured.