Sr gsd

To detect and quantify the interaction between p6^gag and Alix, IFA staining with TIRF and super-resolution fluorescence localization imaging methods were used (Leica SR GSD) [26] (link), [27] for immunostaining, Gag was detected by anti-p24^Gag mouse monoclonal antibody. Alix was detected by anti-Alix rabbit polyclonal antibody. The secondary antibodies were anti-mouse and anti-rabbit fluorescence (Alexa 488 and Alexa 647)-conjugated antibodies. Phosphate buffered saline containing 100 mM β-mercaptoethylamine (MEA) was used for SR fluorescence localization imaging. Imaging fields were magnified using a 100× oil objective (Leica) with a 1.47 numerical aperture and 1.6× optical magnification. The penetration depth of the excitation laser source for TIRF and super-resolution imaging was 200 nm. TIRF fluorescence image stacks consisting of over 30,000 frames were used to calculate SR fluorescence images. A two-dimensional spatial histogram map in each fluorescence channel was calculated using the SR images with an effective pixel size of 20 nm. The co-localization coefficients of two proteins were quantified by a combination of Manders analysis and two-dimensional spatial histogram maps of two fluorescence channels, with the fluorescence background removed during intensity-based co-localization analysis.

Single-molecule localization microscopy was performed as previously described (Moreno et al., 2016 (link)). In brief, we used a ground-state depletion microscope system (SR-GSD, Leica) to generate single-molecule localization maps of Ca_V1.3 and BK channels in tsA-201 cells and neurons. The Leica SR-GSD system was equipped with high-power lasers (488 nm, 1.4 kW/cm²; 532 nm, 2.1 kW/cm²; 642 nm, 2.1 kW/cm²) and an additional 30 mW, 405 nm laser for backpumping. For all experiments, the camera was running in frame-transfer mode at a frame rate of 100 Hz (10 ms exposure time). To restrict the analysis of the fluorescence to the plane of the plasma membrane, the images were acquired in TIRF mode. 25,000–50,000 images were acquired and used to construct localization maps. We estimated a lateral localization accuracy of 16 nm for Alexa-647 (~1900 detected photons per switching cycle). Events with lower counts than 50 (Detection threshold) were treated as noise and discarded.

Frozen sections of kidney tissues were incubated with phosphate buffer saline (PBS) containing 0.1% Triton X-100 (PS0016, Leagene Biotechnology, Beijing, China) for 5 min. The following experimental procedures were conducted according to the TUNEL assay kit (C1090, Beyotime, Shanghai, China). The TUNEL assay solution was prepared (TdT enzyme: fluorescent marker solution = 1:9) and thoroughly mixed. The cell surface was rinsed with PBS once, and each sample was added with 50 μl TUNEL assay solution and incubated at 37°C for 60 min without light exposure. Afterward, the staining solution was removed, and cells were rinsed with PBS three times. After sealing with anti-fluorescence quenching solution, the cells were observed with a fluorescence microscope (Leica SR GSD, Leica, Wetzlar, Germany). According to the distribution of apoptotic cells, five positive visual fields were selected from each section under a light microscope at 200× magnification, and 200 cells were counted in each visual field. The percentage of average apoptotic cells was the apoptotic index.

For super-resolution SMLM-imaging with the Leica SR GSD using the dSTORM/GSDIM protocol, 18 mm coverslips (50000 cells/slide) were stored in PBS after fixation and immunolabelling at 4°C. The coverslips were mounted onto a single depression slide (76 mm ×26 mm) and the cavity filled with 90–100 µl GLOX-MEA buffer (0.5 mg/ml glucose oxidase (Sigma-Aldrich, cat# G7141, 40 μg/ml catalase (Sigma-Aldrich, cat# 02071) 10% w/v glucose (Sigma-Aldrich, cat# 49163), 50 mM Tris-HCl pH 8.0, 10 mM NaCl and 10 mM β-mercaptoethylamine (Sigma-Aldrich, cat# M9768-5G)). The buffer was freshly prepared before imaging. Imaging was performed with a Leica SR GSD system using a HC PL APO 160×/NA 1.43 oil objective. The images were recorded with an Andor iXon 897 EMCCD camera at 40 Hz using a central 180 pixel x 180 pixel subregion. For excitation, a 532 nm laser (500 mW maximum power output) and a 642 nm laser (500 mW maximum power output) were used and attenuated using an AOTF when appropriate. The two fluorophores were recorded sequentially and image acquisition, single molecule analysis and image reconstruction was performed with Leica LAS X 1.9.0.13747.

Frozen sections of the mouse myocardial tissues were incubated in PBS containing 0.1% Triton X-100 for 5 min at ambient temperature. The subsequent steps were performed in strict accordance with the provided instructions of the TUNEL Assay Kit (C1090, Beyotime, Shanghai, China), and the TUNEL assay solution was prepared by combining the TdT enzyme and fluorescent-labeled solution at a ratio of 1 : 9. A total of 50 μL of the TUNEL assay solution was added to each sample. Samples were incubated at 37° C for 60 min in conditions devoid of light. The staining solution was discarded. Droplets of the anti-fluorescence quench were added to the surface and the samples were observed under a fluorescence microscope (Leica SR GSD, Leica, Wetzlar, Germany).