Ab213203

Five-micrometer thick, formalin-fixed, paraffin-embedded lung sections were immunohistochemically analyzed for the expression of HMGB1, forkhead box J1 (FOXJ1), club-cell secretory protein (CCSP), mucin 5AC (MUC5AC), mucin 5B (MUC5B), keratin 5 (KRT5), and KI-67. The sections were stained with the corresponding primary antibodies: rabbit polyclonal HMGB1 antibody (ab18256; Abcam, Cambridge, MA), rabbit monoclonal FOXJ1 antibody (ab235445; Abcam, Cambridge, MA), rabbit monoclonal uteroglobin antibody (ab213203; Abcam, Cambridge, MA), rabbit polyclonal MUC5AC antibody (UNC 294, a kind gift by Dr. Camille Ehre, University of North Carolina, Chapel Hill, NC), rabbit polyclonal MUC5B antibody (UNC223, a kind gift by Dr. Camille Ehre, University of North Carolina, Chapel Hill, NC), rabbit monoclonal Cytokeratin 5 antibody (ab52635; Abcam, Cambridge, MA), and rabbit monoclonal KI-67 antibody (ab16667; Abcam, Cambridge, MA), as published previously (34 (link), 35 (link)).

Paraffin blocks were sliced to a thickness of 4 μm and attached to a silane-coated slide (Muto Pure Chemicals Co., Ltd., Japan). Each procedure was performed according to the ABC kit protocol (Vector Laboratories, USA). After deparaffinization and rehydration, slides were boiled in 0.1 M sodium citrate buffer (pH 6.0) in a microwave oven for antigen retrieval. After cooling the slides to room temperature, 3% H₂O₂ in methanol was used to block the endogenous peroxidase activity. To suppress non-specific reactions, blocking serum (Vector Laboratories) was applied to the tissue. PCNA antibody (Abcam, ab92552, diluted 1:200), prosurfactant protein C (SPC, Abcam, ab90716, diluted 1:1000), and ubiquitin antibody (CC10, Abcam, ab213203, diluted 1:4000) were used as the primary antibodies. Biotinylated antibody (Vector Laboratories, USA) was used as a secondary antibody. It was then detected using the DAB Peroxidase Substrate Kit (Vector Laboratories, USA). All the slides were counterstained with hematoxylin (Gill III hematoxylin, Thermo, USA).

4-hydroxynonenal (4-HNE) is a well-studied aldehyde product of phospholipid peroxidation, indicating the extent of oxidative stress. Clara cell secretory protein (CCSP) is a marker of Clara cells. Both 4-HNE and CCSP measurements were performed by immunofluorescence staining. Briefly, lung tissue slides were deparaffinized, rehydrated, and heated in 0.1 M citrate buffer (pH 5.8) for antigen retrieval. After blocking with 2% BSA for 30 min at room temperature, the slides were incubated with primary antibodies against 4-HNE (#ab48506; Abcam, Cambridge, UK; 1:300) or CCSP (#ab213203; Abcam, Cambridge, UK; 1:2000) overnight at 4°C, followed by Alexa Fluor 488-conjugated IgG second antibody (Thermo Fisher Scientific™, Waltham, MA, USA; 1:1000) and 4’,6-diamidino-2-phenylindole (DAPI) for 1 h at room temperature in the dark. Sections were then observed under a laser scanning confocal microscope (Leica, Germany). F4/80, a marker of mouse mature macrophages, was detected with immunohistochemistry staining as described previously [35 (link)]. All n = 5.