Cf488 tunel cell apoptosis detection kit

The apoptotic cells in the lung tissues were detected by TUNEL staining (CF488 TUNEL Cell Apoptosis Detection Kit, Servicebio) according to the manufacturer’s instructions. Frozen lung sections (4-μm) were defrosted for 20 min at room temperature, permeabilized with 0.2% Triton X-100 for 5 min on ice, and washed twice for 5 min in PBS. Then the samples were stained with TUNEL reaction mixture for 60 min at 37°C in dark, washed twice for 5 min in PBS and mounted with Mounting Medium with DAPI (ab104139, Abcam). The slides were visualized and fluorescent images were captured with fluorescence microscope (ZEISS).

Immunofluorescence staining was performed as previously described (Chen et al., 2020 (link)). In short, the frozen sections were rewarmed at room temperature. After antigen retrieval in citric acid antigen repair solution, the sections were incubated at 4°C overnight with the following primary antibodies: anti-CD31 (1:200, ab222783, Abcam, USA), anti-IBA-1 (1:400, 019-19741, FUJIFILM Wako Shibayagi, Japan), anti-CD68 (1:200, 28058-1-AP, Proteintech, China), anti-GFAP (1:400, #80788, CST, USA), anti-MPO (1:50, A1374, Abclonal, China), and anti-NeuN (1:200, #94403, CST, USA). Then, the sections were incubated with the appropriate fluorescently labeled secondary antibody at room temperature for 1 h. For double staining with TUNEL fluorescence, the procedure followed the instructions of the CF488 TUNEL Cell Apoptosis Detection Kit (G1504, Servicebio, Wuhan, China). After incubation with secondary antibody, the sections were equilibrated with Equilibrium Buffer for 10 min and then incubated at 37°C for 1 h with TDT incubation buffer. Finally, nuclei were stained with DAPI. The sections were observed and photographed with a fluorescence microscope (U-HGLGPS, Olympus, Japan). Micrographs were analyzed with ImageJ software (ImageJ, RRID: SCR _ 003070).

The TUNEL staining procedure was as follows. Briefly, the kidney tissue wax block was dewaxed with xylene and then hydrated with gradient absolute ethanol. Circles were drawn around the tissue with a histochemical pen once the sections dried. Then, the tissue was covered with proteinase K working solution dropwise. After 22 min at 37°C, PBS was used to decolorize the tissue. Following the drying of the sections, 0.1% Triton X-100 solution was poured dropwise in a circle to cover the tissue. The tissue was then incubated for 20 min at room temperature and rinsed three times with PBS. Reaction solution was added to the sections according to the instructions of the CF488 TUNEL Cell Apoptosis Detection Kit (Servicebio, G1501). Slices were incubated with DAPI solution at room temperature for 10 min to counterstain the nucleus. DAPI labeling turned the nucleus blue. Green cells represent positive apoptosis. DAPI emits blue light with a wavelength of 420 nm, and FITC emits green light with an excitation wavelength of 465–495 nm and an emission wavelength of 515–555 nm. Images were collected through a fluorescence microscope. The number of apoptotic cells was counted under the ImageJ software. Five non-overlapping fields of view were randomly selected in each slide to count the number of apoptotic cells.