Alexa flour 555 goat anti rabbit igg

BMDMs were incubated with MitoSOX Red reagent (Thermo Scientific) reagent (1 μM) to examine mitochondrial ROS generation which was visualized by fluorescence microscopy. Cells were also incubated with 10 nM MitoTracker Green (which is insensitive to ROS) to confirm the localization of MitoSOX Red to mitochondria. Immunofluorescence microscopy was carried out to visually identify p62 puncta and lysosomes and to determine co-localization of p62 and lysosomal-associated membrane protein 1 (LAMP1), which indicates autophagosome and lysosome fusion (i.e., activated autophagy). In brief, cells were fixed and permeabilized with cold methanol. Immunocytochemical staining of cells used rabbit anti-p62 monoclonal antibody (Cell Signaling, #23214) or rabbit anti-LAMP1 antibody (Abcam, #ab24210). Alexa Fluor 488 goat anti-rabbit IgG (Thermo Scientific) and Alexa Flour 555 goat anti-rabbit IgG (Thermo Scientific) secondary antibodies were used to detect p62 and LAMP1, respectively. Imaging was acquired via a confocal microscope (Zeiss LSM 880 Confocal with FAST Airyscan).

Cells were grown on 8-well Lab-Tek chamber slides (Thermo Scientific) and fixed 24 h after transfection with paraformaldehyde (4%) in phosphate-buffered saline (PBS) for 10 min at room temperature (RT). Fixed cells were washed with PBS twice and permeabilized with 0.2% Triton X-100 for 5 min at RT, blocked with 5% FCS, and incubated with anti-HA rabbit antibody (1/100) or anti-BALF0/1 rabbit sera (1/200) for 1 h at 37 °C. Then, the cells were washed with PBS and incubated with the secondary antibody at a dilution of 1/1000 (Alexa Flour 555 goat anti-rabbit IgG or Alexa Flour 647 goat anti-rabbit IgG, Thermo Scientific). Next, the cells were washed with PBS and the nuclei were counterstained with Hoechst 33342 (Thermo Scientific). Coverslips were mounted in Glycergel mounting medium (Dako) and observed by using a Zeiss AxioObserver Z1 or Leica SP8 confocal laser microscope. Images were resized, organized, and labeled using ImageJ software. Three-dimensional reconstruction was established by IMARIS (Bitplane, Belfast, UK) software.