Fv3000 confocal laser scanning microscope

EMT6/P and EMT-HER2 cells were labeled Trastuzumab-FITC at 2 µg/mL in PBS with 1% BSA for 30 min at +4 °C. The confocal microscopy images of cells were obtained with an FV3000 laser-scanning confocal microscope (Olympus Optical Co Ltd., Tokyo, Japan) using LUCPLFLN 20× objective (20× magnification, 0.45 numerical aperture) with a 488 nm laser and a GaAsP detector (542 V) (500–600 nm).
The confocal microscopy images of cryosections were obtained with an FV3000 laser-scanning confocal microscope (Olympus Optical Co. Ltd., Tokyo, Japan) using a UPLSAPO 40 × 2 objective (40× magnification, 0.95 numerical aperture) with a 405 nm laser and a GaAsP detector (447 V) (430–470 nm) for Hoechst33342 detection and a 488 nm laser and a GaAsP detector (545 V) (500–600 nm) for FITC detection.

Golgi staining was performed using the FD Rapid GolgiStain Kit (PK401A, FD NeuroTechnologies, Inc.) according to the reported protocol [30 , 31 (link)]. Briefly, whole brains were placed in impregnation solution (a mixture of solution A and solution B) and stored at RT in the dark for 7 days. Then, whole brains were placed in solution C and stored at RT in the dark for 5 days. Next, whole brains were cut coronally at a 200 μm thickness using a VT1200 vibratome (Leica). Brain slices were incubated with staining solution. After rinsing, brain slices were gradually dehydrated with 50%, 75%, 95%, and 100% ethanol. Next, brain slices were cleared with xylenes and mounted with Permount solution (SP15‐100, Thermo Fisher Scientific). For the analysis of dendritic spine density, imaging of secondary dendrites of apical dendrites of pyramidal neurons of II–III cortical layer was performed (z‐stack thickness of 0.5 μm) using an Olympus FV3000 confocal laser scanning microscope (Olympus Life Science) equipped with a UPLSAPO 40 × 2/0.95 N.A. 1.4) objective lens. The number of dendritic spines was determined per micrometer of dendritic length using the ImageJ software (National Instruments of Health).

Harvested at the density of 10 × 10⁶, H9C2 cells were stained with H2DCFDA (MedChemExpress, China) to detect ROS level, and stained with Bodipy (BODIPY™ 581/591 C11, Invitrogen, USA) or Liperfluo (Dojindo, Shanghai, China) to detect lipid peroxidation level. The signals were collected using by flow cytometry (CytoFLEX S equipped with Kaluza analysis 2.1, Beckman coulter, USA) or imaged by an Olympus FV3000 confocal laser scanning microscope (Olympus, Japan).