Image pro plus 3

Tissues were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 24 h at 4°C, washed with PBS, dehydrated by an alcohol gradient and embedded in paraffin. Subsequently, 3 µm sections were sliced, followed by deparaffinization and rehydration through xylene, ethanol and water. The antigen retrieval was performed in 10 mM citrate buffer (pH 6.0) at a constant pressure of 20 cm H₂O. The tissue sections were stained with either hematoxylin and eosin (H&E) for histological analysis, or PAS reagent (G1281) from Beijing Solarbio Science & Technology Co., Ltd., (Beijing, China) for analyzing the content of glycogen; the content of glycogen in alveolar epithelial cells reflect the maturity of AEC II (20 (link)). The sections were observed and images acquired by microscopy at magnification, ×400. PAS staining of the lung tissues revealed red- or purple-colored glycogen. The images were analyzed by Image-Pro Plus III (Media Cybernetics, Inc., Rockville, MD, USA) to obtain the mean gray value. A total of 3–5 samples/group were analyzed.

Cells were fixed with 4% paraformaldehyde for 30 min at room temperature and permeabilized with 0.1% Triton X-100 for 10 min. Subsequently, cells were blocked with 3% bovine serum albumin (Beyotime Institute of Biotechnology) for 2 h at room temperature and incubated with an anti-NDUFA13 primary antibody (1:50; cat. no. ab110240; Abcam) overnight at 4°C. The slides were then washed with phosphate-buffered saline (PBS, Beyotime Institute of Biotechnology) and 0.1% Tween-20 (PBST) and incubated with a Texas Red/Alexa fluor-conjugated secondary antibody for 1 h at room temperature. The slides were mounted using mounting medium, counterstained with 6-diamidino-2-phenylindole (Invitrogen; Thermo Fisher Scientific, Inc.) for 10 min at room temperature and observed using an IX71 microscope (Olympus Corporation; magnification, ×200) and Image Pro Plus 3.0 software (Media Cybernetics, Inc.).