Lsm880 800 confocal microscope

Immunofluorescence staining was performed with anti-CD68 (25747-1-AP, Proteintech, 1:200) using a PANO 4-plex IHC kit (Yuanxi, Shanghai, China) as previously described 19 (link). MEFs were stained with anti-p-STAT3 (Tyr705) (D3A7, cat# 9145, 1:200), anti-p-STAT3 (Tyr727) (cat# 9134, 1:200), anti-HSP60 (D6F1, cat# 12165, 1:1400) using the APExBIO kit (APExBIO, Houston, USA). Anti-p-STAT3 (Tyr705), anti-p-STAT3 (Tyr727) and anti-HSP60 antibodies were purchased from Cell Signaling Technology (MA, USA). The analyses were carried out under a Zeiss LSM880/800 confocal microscope.

For Mito‐Tracker staining, cells treated with 400 nM Mito‐Tracker Red CM‐H2XRos (40740ES50, Yeasen) were fixed and permeabilised. Mitochondrial fragmentation and morphology were analysed by using ImageJ software. For IF staining, the cells were incubated with primary antibodies overnight at 4°C, followed by incubation with the appropriate secondary antibody labelled with Alexa 488 or Alexa 594, as well as DAPI. Finally, analyses were performed using a Zeiss LSM880/800 confocal microscope.

For Mito-Tracker staining, cells were cultured on confocal culture dishes overnight, stained with 400 nM Mito-Tracker Red CM-H2XRos or Mito-Tracker Green FM (40740ES50, 40742ES50, Yeasen) for 30 min at 37 °C, and then washed three times with warm buffer. For MitoSox and Mito-Tracker double staining, cells were stained with Mito-Tracker Green FM and MitoSox for 20 min at 37 °C, and nuclei were then counterstained with Hoechst. The analyses were conducted using a Zeiss LSM880/800 confocal microscope. Mitochondrial fragmentation and morphology were assessed using ImageJ software, and the results were averaged for 10 cells per sample. For double IF staining, the cells were then fixed with 4% paraformaldehyde for 20 min and permeabilized with 0.25% Triton X-100 for 15 min. After the cells were blocked with 5% BSA (Solarbio), they were incubated with the appropriate primary antibodies overnight at 4 °C in PBS containing 1% BSA. After three washes in PBS with Tween 20 (PBST), the cells were incubated with secondary antibodies in 1% BSA for 2 h at 37 °C and rinsed in PBST. Nuclei were then counterstained with DAPI, and analyses were conducted using a Zeiss LSM880/800 confocal microscope.