Cells seeded onto glass coverslips were transfected using Lipofectamine® 2000 (Invitrogen) according to the manufacturer's specifications. 20–24 h post transfection, cells were fixed7 (link) and incubated in anti-SV40 T-ag (Santa Cruz, 1:800) or anti-PHLPP1 (Novus Biologicals, 1:200) or anti-DNMT1 (Abcam, 1:200) primary antibodies, followed by incubation with Alexa 568-labeled goat anti-rabbit secondary antibody or Alexa 568-labeled rabbit anti-mouse secondary antibody (Invitrogen, 1:1000). DNA was counterstained with DNA-specific dye 4,6-diamidino-2-phenylindole (DAPI) using ProLong® Gold Anti-Fade Reagent (Invitrogen).
Immunofluoresence on epididymal sperm was performed using a protocol modified from Herrero et al., 199623 (link). Sperm were fixed with 4% PFA for 10 mins, permeabilized with Triton X-100 (0.1%) and blocked with 1% BSA. Sperm were incubated with anti-BRAP2 (1:200) or anti-AKAP3 (1:100) primary antibody at 4°C overnight, followed by Alexa 568-labeled goat anti-rabbit secondary antibody (Invitrogen, 1:1000) and mounted using ProLong® Gold Antifade with DAPI (Invitrogen Laboratories). Images were captured using a FluoView™ FV1000 confocal microscope, using 100X oil immersion objective lens.
Immunofluoresence on epididymal sperm was performed using a protocol modified from Herrero et al., 199623 (link). Sperm were fixed with 4% PFA for 10 mins, permeabilized with Triton X-100 (0.1%) and blocked with 1% BSA. Sperm were incubated with anti-BRAP2 (1:200) or anti-AKAP3 (1:100) primary antibody at 4°C overnight, followed by Alexa 568-labeled goat anti-rabbit secondary antibody (Invitrogen, 1:1000) and mounted using ProLong® Gold Antifade with DAPI (Invitrogen Laboratories). Images were captured using a FluoView™ FV1000 confocal microscope, using 100X oil immersion objective lens.