Ab180063

Paraffin-embedded fetal lung sections were deparaffinized, rehydrated, and incubated with proteinase K at 37°C for 20 min to retrieve the antigens. The cell membrane was permeabilized with Triton X-100. Apoptotic fetal lung epithelial cells were observed with TUNEL staining (red signals) according to the manufacturer's instructions. For multiplex fluorescent immunohistochemistry, the slides were labeled with LPCAT1 antibody (16112-1-AP, 1:300, Proteintech) or PDPN antibody (ab11936, 1:200, Abcam). Cy5 AffiniPure Goat Anti-Rabbit IgG (GB27301, 1:300, Servicebio) or Goat Anti-Syrian Hamster IgG H&L (ab180063, 1:500, Abcam) was added to slides as a secondary antibody. Then, 100 μl TSA-Cy5 working solution was added to the slides. To determine the number of nuclei, the slides were incubated with DAPI for 10 min at 37°C in the dark and washed with PBS (pH 7.4) three times for 5 min each. Then, the slides were mounted with antifade Fluoromount. Images were obtained using a Nikon Eclipse Ti fluorescence microscope. Quantitative analysis of the TUNEL fluorescence intensity was performed with ImageJ software. Three random fields of view occupied by the fetal lung in each image were defined manually, and the mean fluorescence intensity of the signal within these areas was measured.