Alexa fluor 488 conjugated anti rabbit antibody

BMDMs were seeded on plates seeded with coverslips overnights. BMDMs were then treated as indicated at 37°C. Cells were washed in cold PBS and fixed in cold PFA (4% PFA in PBS) for 15 min at RT. Coverslips were washed 3 times with wash buffer (0.05% tween-20 in PBS) to removed residual PFA. Coverslips were permeabilized for 5 min in 0.1% Triton X-100 in PBS and blocked for 1h at RT in blocking buffer (1% BSA, 0.1% fish gelatin, 0.1% Triton-X-100, 0.05% Tween-20, and 5% donkey serum in PBS). Coverslips were stained with the anti-NFκB or anti-IRF3 antibody (1:250 dilution in blocking buffer) (Cell Signaling; #4764 and #4302), respectively) for 2h at RT. Cells were washed 3X with wash buffer and stained with anti-rabbit-Alexa Fluor 488 conjugated antibody (1 μg/mL) (JacksonImmuno) for 1h at RT. After washing 3X in wash buffer, coverslips were stained with Hoechst 33342 (1:2000) for 10 min at RT. Coverslips were washed 2X in wash buffer prior to mounting with VectaMount medium (Vector Laboratories, CA; #H5000). Mounted samples were cured overnight at RT and imaged within 24h. Images were collected using an Olympus FluoView 1000 under 40X or 60X objective lens. Images were analyzed with ImageJ software.