G1501

Apoptosis of mouse lung tissue was identified using the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) detection kit (G1501, Servicebio, China) and following the manufacturer’s protocol.
The lung tissue of humans or mice was fixed with 4% paraformaldehyde overnight at room temperature, embedded in paraffin, and cut into 3.5 μm sections. Then, sections were incubated with LRG1 (SC-517443, Santa Cruz, USA, 1:50; SC-390920, Santa Cruz, USA, 1:50), KLK10 (bs-2531R, Bioss, China, 1:50), NF-KB (10745-1-AP, proteintech, USA, 1:200), CD31 (ab182981 Abcam, UK, 1:8000) or SPC (GB114059, Servicebio, China, 1:200) at 4^oC overnight. Lung sections were incubated with HRP labeling goat anti-mouse IgG antibody (Servicebio, GB25301, China, 1:400) or HRP labeling goat anti-rabbit IgG antibody (Servicebio, GB21303, China, 1:300) for 50 min at room temperature. Then, the reaction solution was added to the lung tissue, and 4’,6-diamidino-2’-phenylindole (DAPI) (G1012, Servicebio, China) was used to counterstain the nuclei. After mounting, we observed the result under a fluorescence microscope (Nikon Eclipse C1, Japan).

Tissue samples of the uterus were fixed in formalin overnight and embedded in paraffin blocks. The blocks were processed for routine microtome and stained by HE for histopathological observation. Another section was used to assay apoptosis with a fluorescein TUNEL assay kit (G1501, Servicebio Technology Co., Ltd., Hubei, China) based on manufacturer’s instruction. Firstly, tissue sections were deparaffinized and rehydrated. Proteinase K solution was then added to retrieve the antigen. Subsequently, membranes were disrupted, the TUNEL reaction solution was added, and the nuclei were stained with DAPI solution. Finally, microscopic examination and image collection were conducted using a fluorescence microscope (Nikon Instruments Inc., Tokyo, Japan). Blue color indicates cell nucleus, and green color indicates positive apoptosis cells.

The fixed left lung tissues of mice were dehydrated in a series of graded ethanol and embedded in paraffin. The slides of 5 μm thickness were stained with hematoxylin and eosin (H&E) staining for morphometric and histopathological evaluation. The mean linear intercept (MLI) was assessed by qualifying the airspace enlargement and destruction. The MLI was calculated by counting the number of alveolar walls crossed by the reticulum in ten randomly selected fields of tissue sections per lung sample as previously described [35 (link)]. Furthermore, the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) was used to identify apoptotic cells in mice lung tissue by a commercially available kit (Servicebio, G1501, Wuhan, China). The fluorescent staining was examined using the Panoramic MIDI (3DHISTECH Ltd., Budapest, Hungary). The results were expressed as the ratio of the TUNEL-positive cell number to the total number of cells counted.