Alexa fluor 594 conjugated goat anti rabbit igg h l secondary antibody

BMDMs from C57BL/6J mice were grown on coverslips and infected with BCG:gfp followed by treatment with rocaglate for 24 h. Cells were fixed with chilled 100% methanol for 5 min at room temperature and then blocked for 60 min with 1% BSA containing 22.52 mg/mL glycine in PBST (PBS+ 0.1% Tween 20). Cells were incubated with primary antibodies purchased from cell signaling technology [p62(1:200) and LC3B(1:200)] overnight at 4 °C in 1% BSA, and incubated with Alexa Fluor 594-conjugated Goat anti-Rabbit IgG (H+L) secondary Antibody (Invitrogen) in 1% BSA in dark for 1 h. The cells were mounted using Prolong^TM Gold antifade reagent (Thermo Fisher Scientific) and Images were acquired using Zeiss LSM 710-Live Duo scan confocal microscope. All images were processed using ImageJ software.

BMDMs were fixed with 4% paraformaldehyde for 10 min at room temperature and washed twice with 1X PBS. The permeabilization of cell was performed with 0.05% Triton X-100 and then blocked for 60 min with 1% BSA containing 22.52 mg/mL glycine in PBST (PBS+ 0.1% Tween 20). Cells were incubated with primary antibodies (NRF2- or 4-HNE- specific Ab) overnight at 4°C in 1% BSA, washed 3 times with 1X PBS for 5 min, and incubated with Alexa Fluor 594-conjugated Goat anti-Rabbit IgG (H+L) secondary Antibody (Invitrogen) in 1% BSA in dark for 1 h. The cells were mounted using ProlongTM Gold antifade reagent (Thermo Fisher Scientific) and Images were acquired using Leica SP5 confocal microscope. All images were processed using ImageJ software.
Alternatively, cells were plated in 96 well glass bottom plate (PerkinElmer) and processed and stained as above. After secondary antibody incubation cells were directly imaged and analyzed using Operetta CLS High Content Analysis System (PerkinElmer).