Immunofluorescent analysis of PAX6 (green) in All-Trans-Retinoic Acid (ATRA) treated (10 µM for 24 hours) NCCIT cells. Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 1% BSA for 15 minutes at room temperature. Cells were probed with a PAX6 polyclonal antibody(ab5790) at a dilution of 1:50 for at least 1 hour at room temperature and incubated with DyLight 488 goat-anti-rabbit IgG secondary antibody at a dilution of 1:400 for 30 minutes at room temperature. F-Actin (red) was stained with DyLight 554 Phalloidin and nuclei (blue) were stained with Hoechst 33342 dye. Images were taken at 20X magnification.

Western blot analysis of PAX6 was performed by loading 50ug of various Human, mouse and non-human primate whole cell lysates per well onto a 4-20% Tris-HCl polyacrylamide gel. Proteins were transferred to a PVDF membrane and blocked with 5% BSA/TBST for at least 1 hour. The membrane was probed with a PAX6 polyclonal antibody (ab5790) at a dilution of 1:1000 overnight at 4°C on a rocking platform then washed in TBS-0.1%Tween 20 and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:20000 for at least one hour. Chemiluminescent detection was performed.

Immunoprecipitation of PAX6 was performed on 293T cells. Antigen: antibody complexes were formed by incubating 500 µg whole cell lysate with 5 µg of PAX6 polyclonal antibody (ab5790) overnight on a rocking platform at 4°C. The immune complexes were captured on 50 µl Protein A/G Agarose, washed extensively and eluted with 5X Buffer. Samples were resolved on a 4-20% Tris-HCl polyacrylamide gel then transferred to a PVDF membraneand blocked with 5% BSA/TBS-0.1%Tween for at least 1 hour. The membrane was probed with a PAX6 monoclonal antibody at a dilution of 1:1000 overnight rotating at 4°C then washed in TBST and probed with detection reagent at a dilution of 1:1000 for at least one hour. Chemiluminescent detection was performed.

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