Histopathological and immunohistochemical studies

Histopathological and immunohistochemical procedures were performed as described elsewhere.²³ Briefly, rat lungs were inflated at 20 cm H₂O with 0.05% (w/v) agarose in 1% neutral buffered formalin and fixed in 10% neutral buffered formalin. Formalin-fixed lungs were paraffin embedded, and 5-µm-thick tissue sections were prepared. For histopathological analysis, the tissue was stained with hematoxylin and eosin.

For immunohistochemical staining of human and rat lung sections, FFPE sections were deparaffinized and rehydrated. Endogenous peroxide activity was blocked with hydrogen peroxide for 10 minutes. For antibody epitope retrieval, heated citrate buffer was applied for 30 minutes. The sections were then blocked with normal horse serum for 1 hour. Tissue sections were incubated overnight at 4°C with primary antibodies against SphK1 and SphK2 (Abgent; 1∶500). Sections were incubated with biotinylated secondary antibodies for 30 minutes, washed with phosphate-buffered saline, and incubated in ABC Reagent (Vector Labs) for another 30 minutes. Western blot analysis was performed to determine antibody selectivity. Rabbit immunoglobulin G isotype was used as a negative control. Diaminobenzidine, as a substrate for horseradish peroxidase, was used to identify antibody-labeled cells. Hematoxylin was used as a counterstain. Lung sections were analyzed by light microscopy.