Lsm 880 with airyscan

Wide-field fluorescence imaging was performed on inverted microscopes (IX83, Olympus) using an oil-immersion PlanApo 60× NA 1.42 objective lens. Digital still images were captured by ORCA-Flash4.0 LT+ digital CMOS camera (Hamamatsu) through the software MicroManager (Open Imaging) (62 (link)). For imaging of membrane infoldings, multiple z-stack images were captured on a confocal microscope (LSM 880 with Airyscan, Carl Zeiss) using an oil-immersion 63× NA 1.4 DIC objective lens. Images were captured with 32-channel GaAsp photomultiplier modules using ZEN 2.3 software (Carl Zeiss). All confocal z-stack images were subjected to Airyscan processing. For imaging and tracking of AChR vesicles, high-speed time-lapse images were captured at 200 ms per frame on live-SR super-resolution module on an iLas3 Ring-TIRF system (Gataca Systems). All acquisition settings (i.e., laser power and gain) were kept the same for different experimental groups in the same experiment. All acquired images were analyzed by ImageJ (NIH) or Imaris (Bitplane).

Kidney tissues from control or STZ-induced diabetic mice treated with or without APX-115 or losartan, were embedded as frozen block. DHE (5 µM, Molecular Probes) was applied to the freshly cut frozen kidney segments (10 µm) for 10 min at 37°C to reveal the presence of ROS with red fluorescence at 561 nm followed by DAPI staining and imaging by LSM880 with airy scan (Carl Zeiss Microscopy GmbH, 07745 Jena, Germany). Fluorescence intensities were then quantified using Image-Pro Plus 4.5 software (Media 149 Cybernetics, Silver Springs, MD, USA).

After serum starvation, mesangial cells were incubated either 5.5 mM glucose or 30 mM glucose for 24 h and then angII (1 µM, Sigma, St. Louis, MO, USA) for 30 min. To see the effect of APX-115, 1 µM APX-115 was treated for 30 min before treating high glucose. The cells were then washed with Hanks’ balanced salt solution (HBSS) and incubated for 10 min in the dark at 37°C HBSS containing 10 µM DCF-DA (Molecular probes). Fluorescence of oxidized DCF was detected using Zeiss LSM880 airy scan (Carl Zeiss Microscopy GmbH, 07745 Jena, Germany) at excitation wavelengths of 488 nm. Four fields of each cell were randomly selected and the fluorescence intensity was measured with a Zeiss vision system (LSM880 with airy scan) and the mean relative fluorescence intensity was measured by the average of random four values.

Immunofluorescence staining was performed on different days with behavior tests. The procedures were described in previous work [24 (link)]. Briefly, 2 h after the acute drug injection, the mice were anesthetized with tribromoethanol (20 mg/kg), perfused with phosphate-buffered saline (PBS), and 4% paraformaldehyde (AR1068; Boster Biological Technology, Wuhan, China) for pre-fixation. The brain tissue was removed, placed in 4% paraformaldehyde, and stored at 4 °C for 24 h, then dehydrated with 20% and 30% sucrose solutions for 2 days. The OTC-embedded tissue was cut into 40-μm sections using a freezing microtome (CM1950; Leica, Wetzlar, Germany). After permeabilization and blocking, the sections were incubated with primary anti-c-fos antibody (1:500, rabbit, #2250; Cell Signaling Technology, Danvers, USA) at 4 °C for 20 h and then washed three times with PBS. Afterward, the sections were incubated with the secondary Alexa Fluor488-conjugated donkey anti-rabbit antibody (1:500, A21208; Invitrogen, Carlsbad, USA) at room temperature for about 2 h and washed with PBS. To stain the nuclei, 4′,6′-diamidino-2-phenylindole (DAPI, 0.1 μl/ml) was used for about 5 min. Sections were washed with PBS and covered on a glass slide. Brain slices were imaged using a confocal microscope (LSM 880 with Airyscan; Zeiss, Jena, Germany).